Vaccination of immunocompromised subjects

ABSTRACT

Disclosed herein are methods for enhancing immune responses to a vaccine in immunocompromised individuals, including those receiving a statin therapy. Related products are also provided.

FIELD OF THE INVENTION

The present disclosure generally relates to enhancing an immune responsein individuals immunocompromised by medications or for other reasons.

BACKGROUND OF THE INVENTION

Immunosuppression induced by drugs or other reasons in a subject is anobstacle to achieving effective vaccination of the subject. It isimportant to note that individuals may be immunocompromised for avariety of reasons, including, for example, disease or disorderassociated with immunosuppression, age (e.g., the elderly), and being onmedications or medical procedures that suppress or otherwise interferewith their immune response.

Certain drugs have been implicated in causing immunomodulation inpatients, including statins, non-steroidal anti-inflammatory drugs(NSAIDs), interferons, and certain antipsychotic drugs, such asclozapine and haloperidol. Such immunomodulatory effects include adverseor unwanted immunosuppression in the patients, particularly those on along-term therapeutic regimen.

Statins are a class of drugs used to lower cholesterol levels byinhibiting the enzyme HMG-CoA reductase. Because of the associationbetween elevated cholesterol levels and the risk of cardiovasculardisease and because of studies showing that statins can lower this risk,statins have been given to large numbers of individuals, mostly olderadults (Lewington S, Whitlock G, Clarke R, Sherliker P, Emberson J,Halsey J, Qizilbash N, Peto R, Collins R. (2007) “Blood cholesterol andvascular mortality by age, sex, and blood pressure: a meta-analysis ofindividual data from 61 prospective studies with 55,000 vasculardeaths.” Lancet 370 (9602): 1829-39). Although the primary goal ofstatin therapy has been to lower cholesterol, it has been recognizedthat this drug class has other effects including immunomodulatory andanti-inflammatory effects (Jain, M. K., & Ridker, P. M. (2005)“Anti-inflammatory effects of statins: clinical evidence and basicmechanisms.” Nature Reviews Drug Discovery, 4(12): 977-987).

Secondary effects of a statin therapy on individuals, including bothelderly and non-elderly populations, have been somewhat controversial inthe literature, although most studies have concluded that statin canelicit immunomodulatory effects and that such effects are complex.

Non-steroidal anti-inflammatory drugs (NSAIDs) are a class of drugs thatprovides analgesic (pain-killing) and antipyretic (fever-reducing)effects, and, in higher doses, anti-inflammatory effects. NSAIDs aresometimes also referred to as nonsteroidal anti-inflammatoryagents/analgesics (NSAIAs) or nonsteroidal anti-inflammatory medicines(NSAIMs). Due to the importance of pain management, the use of NSAIDshas increased dramatically in recent decades. However, all NSAIDs havethe potential for certain adverse effects, including potentiallyunwanted suppression of an immune response.

Interferons (IFNs) are a group of signaling proteins made and releasedby host cells in response to the presence of pathogens, such as viruses,bacteria, parasites, or tumor cells. In a typical scenario, avirus-infected cell will release interferons causing nearby cells toheighten their anti-viral defenses. Interferon beta-1a and interferonbeta-1b are used to treat and control multiple sclerosis, an autoimmunedisorder. This treatment is effective for reducing attacks inrelapsing-remitting multiple sclerosis and slowing disease progressionand activity in secondary progressive multiple sclerosis.

The most frequent adverse effects reported by patients receivinginterferon therapy are flu-like symptoms: increased body temperature,feeling ill, fatigue, headache, muscle pain, convulsion, dizziness, hairthinning, and depression. Erythema, pain and hardness on the spot ofinjection are also frequently observed. IFN therapy causesimmunosuppression, in particular through neutropenia and can result insome infections manifesting in unusual ways. IFN therapy may alsoexhibit increased susceptibility to secondary infections following aviral infection, such as influenza. In such cases, the patientco-infected with the primary pathogen (such as influenza virus) and asecondary pathogen (such as a bacterial infection) may be at elevatedrisk of developing complication from the secondary infection.

Certain antipsychotic compounds have been reported to cause side effectsincluding immunosuppression in subjects.

Interferon therapy is used (in combination with chemotherapy andradiation) as a treatment for some cancers.[26] This treatment can beused for treating hematological malignancy; leukemia and lymphomasincluding hairy cell leukemia, chronic myeloid leukemia, nodularlymphoma, and cutaneous T-cell lymphoma.[26] Patients with recurrentmelanomas receive recombinant IFN-α2b.[27] Both hepatitis B andhepatitis C are treated with IFN-α, often in combination with otherantiviral drugs.[28][29] Some of those treated with interferon have asustained virological response and can eliminate hepatitis virus. Themost harmful strain—hepatitis C genotype I virus—can be treated with a60-80% success rate with the current standard-of-care treatment ofinterferon-α, ribavirin and recently approved protease inhibitors suchas Telaprevir (Incivek) May 2011, Boceprevir (Victrelis) May 2011 or thenucleotide analog polymerase inhibitor Sofosbuvir (Sovaldi) December2013 [30]. Biopsies of patients given the treatment show reductions inliver damage and cirrhosis. Some evidence shows giving interferonimmediately following infection can prevent chronic hepatitis C,although diagnosis early in infection is difficult since physicalsymptoms are sparse in early hepatitis C infection. Control of chronichepatitis C by IFN is associated with reduced hepatocellularcarcinoma.[31]

Interferon treatment was evaluated in individuals suffering from herpessimplex virus epithelial keratitis. Topical interferon therapy was shownto be an effective treatment, especially with higher concentrations.[32]Interferon, either used alone or in combination with debridement,appears to be as effective as a nucleoside antiviral agent.[32] Thecombination of interferon and another nucleoside antiviral agent mayspeed the healing process.[32]

When used in the systemic therapy, IFNs are mostly administered by anintramuscular injection. The injection of IFNs in the muscle or underskin is generally well tolerated. The most frequent adverse effects areflu-like symptoms: increased body temperature, feeling ill, fatigue,headache, muscle pain, convulsion, dizziness, hair thinning, anddepression. Erythema, pain and hardness on the spot of injection arealso frequently observed. IFN therapy causes immunosuppression, inparticular through neutropenia and can result in some infectionsmanifesting in unusual ways.[33]

To date, there has been no consensus in the art as to the interplaybetween the pharmacokinetics of these drugs and their modulatory effectson various aspects of immune function in vivo and how such effectsshould be taken into consideration in the overall management of healthin patients.

SUMMARY OF THE INVENTION

It has now been found that certain vaccine compositions and/or vaccineregimens can be effectively used to enhance a desirable immune responsein an individual whose immune system is compromised. In particular, thepresent invention includes the recognition that a patient population oncertain therapies, such as statin therapy, shows reduced immunogenicityto a vaccine, and that the use of an adjuvanted vaccine (e.g., vaccinesformulated with an oil-in-water adjuvant) and/or a high-dose antigen mayrestore or even enhance an immune response to such a vaccine.

In a broad sense, the present invention describes immunization of asubject whose immune system is compromised for one or more reasons.Accordingly, the invention provides methods for immunizing certaintarget populations, including recipients of an immunomodulatory therapy(e.g., medications), such as statin therapy, NSAID therapy, interferontherapy, and/or antipsychotics therapy, by administration of a vaccinecomposition that is (i) adjuvanted, and/or (ii) containing a high doseantigen, to the subject in an effective amount, so that, as compared toan equivalent unadjuvanted or standard-dose vaccine, the subject elicitsa better immune response to the same antigen(s) contained in thevaccine. According to the invention, the methods described herein mayprovide particularly beneficial immune protection to those subjects whodo not otherwise produce a desired immune response to a vaccine due tomedications that cause adverse immunosuppression.

Accordingly, the present invention is suitable for vaccinating subjectsconsidered to be “at-risk.” In some embodiments, the at-risk criteriainclude but are not limited to individuals with confirmed medicalhistory of any of the following: endocrine disorders, chroniccardiovascular diseases, chronic pulmonary diseases, chronic renal orhepatic diseases, neurological and neurodevelopmental conditions, blooddisorders, metabolic disorders, weakened immune system, obesity, receiptof long term aspirin therapy and/or any of the therapies listed above.In some embodiments, at-risk subjects are characterized in that they aremore susceptible to developing secondary infection following a primaryinfection and that they are at higher risk of developing complicationsdue to the co-infections (i.e., the primary and the secondaryinfections). Without wishing to be bound by a particular theory, it iscontemplated that a primary infection, such as influenza infection, mayrender the person more susceptible to a secondary infection due toimmunosuppression. As a result, the subject may develop the secondaryinfection that is atypically severe. In some embodiments, the secondaryinfection is a bacterial infection, e.g., respiratory infection, skininfection, etc. The invention thus aims to counter suchimmunosuppressive effects by the use of protective influenza vaccinescomprising an adjuvant, high dose antigens, or combination thereof,which in turn may boost the subjects' immune system to fight against thesecondary infection more effectively. Thus, in some embodiments, betterprotection against an influenza infection in an at-risk subject provideslesser probability of the subject developing complication from asecondary infection.

In some embodiments, the invention provides methods for administering aninfluenza vaccine to an at-risk subject in an amount effective to elicita protective immune response to the vaccine antigen(s). Preferably, suchinfluenza vaccine is an adjuvanted vaccine. In situations where asubject receives multiple doses of influenza immunization (e.g., apriming dose and a booster dose), it is possible to administer anadjuvanted influenza vaccine in one dose or more, in any order. Forexample, a priming dose may be unadjuvanted, while a subsequent boosterdose may be adjuvanted, or vice versa.

In some embodiments, the invention provides methods for administering aninfluenza vaccine to a subject receiving an immunosuppression-causingtherapy (e.g., a statin therapy, an NSAID therapy, interferon therapy,antipsychotics therapy, etc.) in an amount effective to elicit aprotective immune response to the vaccine antigen(s). Preferably, suchinfluenza vaccine is an adjuvanted vaccine. In situations where asubject receives multiple doses of influenza immunization (e.g., apriming dose and a booster dose), it is possible to administer anadjuvanted influenza vaccine in one dose or more, in any order. Forexample, a priming dose may be unadjuvanted, while a subsequent boosterdose may be adjuvanted, or vice versa.

The invention further provides related vaccine compositions for use in amethod for enhancing an immune response in subjects, including those whoare on an immunosuppression-causing therapy (e.g., a statin therapy, anNSAID therapy, interferon therapy, antipsychotics therapy, etc.). Insome embodiments of the invention, such vaccine compositions areformulated with an adjuvant. Preferred adjuvants include oil-in-wateremulsion-based adjuvants, such as those comprising squalene.Additionally or alternatively, vaccine compositions of the presentinvention may comprise a high-dose antigen, a standard-dose antigen, ora low-dose antigen. Related kits are also described.

The invention also provides a flu vaccine for a statin-treated patient,in particular an adjuvanted or high-dose flu vaccine for astatin-treated patient, preferably a patient treated with a syntheticstatin. In some embodiments, the patient is 65 years or older. Inalternative embodiments, the patient is under 65, but 18 years or older(i.e., between the age of 18 and 64).

The invention includes a flu vaccine for use in a statin-treatedpatient, in particular an adjuvanted or high dose flu vaccine for use ina statin-treated patient, preferably a patient treated with a syntheticstatin. In some embodiments, the patient is 65 years or older. Inalternative embodiments, the patient is under 65, but 18 years or older(i.e., between the age of 18 and 64).

The invention further includes a flu vaccine for prevention of flu in anat-risk subject, such as a statin-treated patient, in particular anadjuvanted or high dose flu vaccine for prevention of flu in astatin-treated patient, preferably a patient treated with a syntheticstatin. In some embodiments, the patient is 65 years or older. Inalternative embodiments, the patient is below 65, but 18 years or older(i.e., between the age of 18 and 64).

The invention also provides a composition of the invention for use as amedicament, and provides the use of a composition of the invention forthe manufacture of a medicament for raising an immune response in asubject who is statin-treated, preferably a patient treated with asynthetic statin. In some embodiments, the patient is 65 years or older.In alternative embodiments, the patient is under 65, but 18 years orolder (i.e., between the age of 18 and 64).

The invention further provides a method for manufacturing an adjuvantedor high-dose flu vaccine, whereby the following steps are conducted: aflu virus is grown in eggs or in a suitable cell line; the virus isharvested and purified, optionally the virus is split and the antigensare isolated; optionally the virus or the antigens are formulated andfilled as a final vaccines, whereby the vaccine is for use in astatin-treated patient, preferably a patient treated with a syntheticstatin. In some embodiments, the patient is 65 years or older. Inalternative embodiments, the patient is under 65, but 18 years or older(i.e., between the age of 18 and 64).

When adjuvants are used in combination with flu antigens herein, theantigen component and the adjuvant component of the vaccine may bepremixed, or alternatively, may be presented in separate containers formixing by the end-user/health care provider before administration. Theantigen component and the adjuvant component may be produced in the sameproduction site or in different production sites. One aspect of theinvention is the use of the antigen component(s) for formulation and/orpackaging into a kit with an adjuvant component, wherein the kit is foruse in patients who are statin-treated, preferably patients treated witha synthetic statin. Another aspect of the invention is the use of theadjuvant component for formulation and/or packaging into a kit with anantigen component for use in at-risk patients, such as those who arestatin-treated, preferably patients treated with a synthetic statin. Insome embodiments, the patient is 65 years or older. In alternativeembodiments, the patient is below 65, but 18 years or older (i.e.,between the age of 18 and 64).

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

A number of factors can cause impairment of one or more aspects ofimmune function in a subject, rendering the subject to beimmunocompromised. Certain medications, such as statins, NSAIDs,interferons, and antipsychotics, are known to cause unwanted immunemodulations. In the context of the present disclosure, the phrase “animmunosuppression-causing therapy” refers to a medical intervention ortreatment that is associated with side effects characterized by unwantedimmunosuppressive effects. Non-limiting examples of such therapiesinclude a statin therapy, an NSAID therapy, interferon therapy,antipsychotics therapy, etc.

Statins are a class of drugs used to treat hypercholesterolemia and arefrequently used to reduce the risk of cardiovascular disease. However,statins have been reported to exert certain immunomodulatory effectswhich could impact vaccine response in those who are on a statintherapy, including influenza vaccines. Similarly, other drugs widelyadministered for treating or alleviating certain conditions are alsoassociated with immunosuppressive side effects.

As further detailed herein, immunogenicity measurements of adjuvantedversus unadjuvanted influenza vaccine obtained from individuals who areeither on or off statin therapy have revealed significantimmunosuppressive effects of statin in those receiving statin, ascompared to control individuals not receiving statin therapy. As furtherdemonstrated below (see the EXEMPLIFICATION section below), thisimmunosuppressive effect of statins on vaccine immune response isparticularly dramatic in individuals on synthetic statins. These effectsare seen in both the adjuvanted and unadjuvanted vaccine groups.Strikingly, however, the negative impact of statin therapy on a vaccineresponse can be at least partially counteracted by the use of anadjuvanted vaccine and/or a high-dose antigen or antigens.

Accordingly, the invention provides methods for enhancing an immuneresponse to a vaccine in a subject whose immune system is compromised,e.g., immunosuppressed or at risk of developing immunosuppression. Suchmethods comprise administration of a vaccine composition that either (i)is adjuvanted, (ii) contains one or more high-dose antigens, to suchsubjects in an amount effective to enhance their immune responses to thevaccine.

Immunomodulatory Therapy

As used herein, “an immunomodulatory therapy” refers to any medicalinterventions, such as medications, that cause modulations in an immuneresponse in patients. Such modulatory effects include immunosuppression,which can cause reduced immune response to a vaccine, leaving therecipient of the therapy more susceptible to an infection or relateddisease. Examples of immunomodulatory therapies include statins,non-steroidal anti-inflammatory drugs (NSAIDs), interferons,antipsychotics, etc.

Statins

As used herein, statins refer to a class of drugs generally known asHMG-CoA reductase inhibitors and also encompass other compounds havingequivalent biological activities (i.e., the ability to inhibit HMG-CoAreductase activities). Statins referred to in the present disclosure maybe non-synthetic (e.g., fermentation-derived or naturally occurring) orsynthetic. Non-limiting examples of non-synthetic statins include:Pravastatin, Simvastatin, Lovastatin and Mevastatin. Non-limitingexamples of synthetic statins include: Fluvastatin, Atorvastatin,Cerivastatin, Rosuvastatin and Pitavastatin. Any statins may be used incombination, either administered simultaneously or separately but inconjunction with each other. Statins may also be administered ascombination formulations that include at least one statin and at leastone non-statin compound. Examples of statin-containing products include,but are not limited to: ADVICOR® (niacin extended-release andlovastatin), CADUET® (amlodipine besylate/atorvastatin calcium),VYTORIN® (simvastatin and ezetimibe) and SIMCOR® (simvastatin niacinextended release).

A “statin therapy” therefore refers to a regimen that includes one ormore statins either alone or in any combination, regardless of aparticular condition, for which the statin or statins are beingadministered. In some embodiments, a statin therapy includes at leastone synthetic statin.

NSAIDs

Nonsteroidal anti-inflammatory drugs, usually abbreviated to NSAIDs, area class of drugs that provides analgesic (pain-killing) and antipyretic(fever-reducing) effects, and, in higher doses, anti-inflammatoryeffects. NSAIDs can be classified based on their chemical structure ormechanism of action. Typically, older NSAIDs were known long beforetheir mechanism of action was elucidated and were for this reasonclassified by chemical structure or origin. Newer substances, on theother hand, are more often classified by mechanism of action.

Non-limiting examples of NSAIDs include: Salicylates (e.g., Aspirin(acetylsalicylic acid), Diflunisal (Dolobid™), Salsalate (Disalcid™) andCholine Magnesium Trisalicylate (Trilisate™)); Propionic acidderivatives (e.g., Ibuprofen, Dexibuprofen, Naproxen, Fenoprofen,Ketoprofen, Dexketoprofen, Flurbiprofen, Oxaprozin and Loxoprofen);Acetic acid derivatives (e.g., Indomethacin, Tolmetin, Sulindac,Etodolac, Ketorolac, Diclofenac, Aceclofenac and Nabumetone); Enolicacid (Oxicam) derivatives (e.g., Piroxicam, Meloxicam, Tenoxicam,Droxicam, Lornoxicam and Isoxicam); Anthranilic acid derivatives(Fenamates) (e.g., Mefenamic acid, Meclofenamic acid, Flufenamic acidand Tolfenamic acid); Selective COX-2 inhibitors (Coxibs) (e.g.,Celecoxib, Rofecoxib, Valdecoxib, Parecoxib, Lumiracoxib, Etoricoxib andFirocoxib); Sulfonanilides (e.g., Nimesulide) and others, such asLicofelone, H-harpagide and Lysine clonixinate.

Interferons

Non-limiting examples of therapeutic interferons available in the marketinclude: Interferon alpha 2a (e.g., Roferon A); Interferon alpha 2b(Intron A/Reliferon/Uniferon); Human leukocyte Interferon-alpha(HuIFN-alpha-Le) (Multiferon); Interferon beta 1a, liquid form (Rebif);Interferon beta 1a, lyophilized (Avonex); Interferon beta 1a, biogeneric(Iran) (Cinnovex); Interferon beta 1b (Betaseron/Betaferon); Interferongamma 1b (Actimmune); PEGylated interferon alpha 2a (Pegasys); PEGylatedinterferon alpha 2a (Egypt) (Reiferon Retard); PEGylated interferonalpha 2b (PegIntron); and PEGylated interferon alpha 2b plus ribavirin(Canada) (Pegetron).

Antipsychotics and Antidepressants

Non-limiting examples of antipsychotics and/or antidepressants that maycause immunosuppression include: clozapine, haloperidol, as well asinhibitors of serotonin receptors and/or dopamine receptors. Additionalexamples include but are not limited to: SB-258719 (a neutral 5HT7Rantagonist available from GSK), SB-258741 (“AFZ”; a partial inverse5HT7R agonist available from GSK), SB-269970 (a robust 5HT7R inverseagonist available from GSK), Risperidone (an antagonist of the D1 and D2dopamine receptors and an inverse agonist of the 5HT7 serotoninreceptors; also an inverse agonist of H1 and H2 histamine receptors),Sertindole (binds to D2, 5-HT2A, 5-HT2C, 5-HT6, 5-HT7, D3, a1);Ziprasidone (binds to D2, 5-HT2A, 5-HT1A, 5-HT1D, 5-HT2C, 5-HT7, D3, a1,NRI, SRI); Loxapine (binds to D2, 5-HT2A, 5-HT6, 5-HT7, D1, D4, a1, M1,H1, NRI); Zotepine (binds to D2, 5-HT2A, 5-HT2C, 5-HT6, 5-HT7, D1, D3,D4, a1, H1, NRI); Clozapine (binds to D2, 5-HT2A, 5-HT1A, 5-HT3, 5-HT6,5-HT7, D1, D3, D4, a1, a2, M1, H1); Olanzapine (binds to D2, 5-HT2A,5-HT2C, 5-HT3, 5-HT6, D1, D3, D4, D5, a1, M1-5, H1), Quetiapine (bindsto D2, 5-HT2A, 5-HT6, 5-HT7, a1, a2, H1); Promethazine (a strongantagonist of the H1 histamine receptor with weak to moderate affinityfor the 5HT2a/c serotonin receptors and dopamine D2 receptor; also ablocker of Na+ channels). Certain ion channel blockers may be alsosuitable. Examples include, but are not limited to: inhibitors andblockers of voltage-dependent Na+ channels and cholinesterase, involvedin lipid transport and metabolism, such as Dibucaine (a butynesteraseinhibitor); inhibitors and blockers of voltage-gated L-type calciumchannels (such as Nimodipine); inhibitors and blockers of sodiumchannels such as Aprindine (a Class 1b antiarrhythmic membranestabilizing agent), Amiloride (a direct blocker of epithelial sodiumchannel ENaC); and inhibitors and blockers of delayed inward rectifierpotassium channels and L-type calcium channels such as Ibutilidehemifumarate (a Class III antiarrhythmic agent)

Subjects

The terms “subject,” “patient” and “individual” may be usedinterchangeably herein. As already alluded to, the methods andcompositions described herein are useful for immunizingimmunocompromised subjects or those at risk of developingimmunosuppression. Described methods and compositions are particularlyuseful for eliciting sufficient immunoprotection in those who areimmunosuppressed or at risk of developing immunosuppression due tocertain medications that may render the subject vulnerable to infection.

Thus, in the context of the invention, suitable target populationsinclude subjects on certain therapies, long-term therapies inparticular. These include subjects on a statin therapy, regardless ofthe age of the subject, including a long-term use of statin. These alsoinclude subjects on an NSAID therapy, regardless of the age of thesubject, including a long-term use of NSAID. Target populations alsoinclude subjects on more than one such therapies. For example, a subjectmay be on one or more statin therapies, one or more NSAID therapies, orcombination of both.

When an individual's immune system is not functioning properly, theindividual is generally said to be “immunocompromised.” Thus, “animmunocompromised subject” is a subject with reduced ability to elicitan appropriate immune response due to any host-related, medical-relatedor pharmacological related factor. An immunocompromised individual mayexhibit one or more types of impairment of the immune system, such asimmunosuppression (e.g., weakened immunity), immunodeficiency, alteredor overactive immune system, autoimmunity, or any combination thereof.Thus, an immunocompromised individual is not fully immune-competent.

In some population groups, subjects may be immunocompromised as part ofnormal development such as age-related conditions, not related to aspecific disease or disorder. For example, very young children (e.g.,infants) and the elderly may be considered not fully immune-competent.According to the invention, a target population of particular interestincludes individuals who are currently on an immunomodulatory therapy(e.g., taking statin, NSAID, etc.) who have recently received animmunomodulatory therapy (e.g., taking statin, NSAID, etc.), as well asthose who are about to or scheduled to start a therapy that includes animmunomodulatory therapy (e.g., taking statin, NSAID, etc.), e.g., asynthetic statin. Any of such subpopulations of patients may becollectively said to be “on immunomodulatory therapy,” for example, “onstatin therapy.” In some embodiments, a target population comprises orconsists of individuals on at least one synthetic statin.

In some embodiments, a subject has been (and continues to be) on animmunomodulatory therapy (e.g., a statin therapy, an NSAID therapy,etc.) for at least 2 days, at least 3 days, at least 4 days, at least 5days, at least 6 days, at least 7 days, at least 10 days, at least 2weeks, at least 3 weeks, at least 4 weeks, at least 6 weeks, at least 8weeks, at least 12 weeks, or longer. In some embodiments, a subject waspreviously on an immunomodulatory therapy (e.g., a statin therapy, anNSAID therapy, etc.) as above, but has ceased such therapy within thelast 6 weeks, 5 weeks, 4 weeks, 3 weeks, 2 weeks, 1 week, 6 days, 5days, 4 days, 3 days or 2 days. In yet other embodiments, a subject hasnot yet been on an immunomodulatory therapy (e.g., a statin therapy, anNSAID therapy, etc.) but is scheduled to or is about to begin such atherapy within the next day, 2 days, 3 days, 4 days, 5 days, 6 days, 7days, 10 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks or 6 weeks. Along-term or chronic use of an immunomodulatory therapy (e.g., a statintherapy, an NSAID therapy, etc.) may refer to a duration of at least 4weeks, inclusive.

In the context of the present disclosure, it shall be understood that asubject is “at risk of immunosuppression” if the subject has propensityfor developing a condition that includes suppressed immune responses orif the subject meets certain criteria for a population associated withheightened risk of immunosuppression or susceptible to having acompromised immune system. The term “susceptible” means having anincreased risk for and/or a propensity for (typically based on age,genetic predisposition, environmental factors, personal history, orcombinations thereof) something, i.e., a disease, disorder, or condition(such as, for example, compromised immune response) than is observed inthe general population. For example, a subject who is about to start onan immunomodulatory therapy (e.g., a statin therapy, an NSAID therapy,etc.) is at risk of immunosuppression and thus may benefit from themethods described herein in order to boost an immune response to avaccine.

Subjects may be selected on the basis of certain criteria (i.e., atarget population). As an example, among statin recipients, a populationof individuals associated with at least one synthetic statins may be athigher risk of immunosuppression than those associated withnon-synthetic statins.

Certain disease and disorders are known to be associated with impairedimmunity in affected individuals. Impaired immunity may involve asuppressed immune system, an overactive immune system, autoimmunity, orany combination thereof. Immunosuppression includes, without limitation,primary immune deficiency and secondary or acquired immune deficiency.Primary immune deficiency may be caused by genetic abnormality.Secondary or acquired immune deficiency is commonly associated withcertain disease, such as AIDS, as well as temporary acquired immunedeficiencies due to certain drugs, such as statins NSAIDs,chemotherapeutics or immunosuppressing agents administered followingorgan transplants. One's immune system can also be weakened by certainliving conditions or environmental factors, including smoking, alcoholconsumption and poor nutrition.

For example, a target population may consist of subjects of one agegroup, such as infants and elderly subjects. As used herein, an “elderlysubject” is a human individual of age 65 or older. An “infant,” is ahuman individual between the age of 0-12 months (0 being a newborn), forexample, between 0-3 months, between 0-6 months, between 0-9 months, andbetween 6-12 months. In some embodiments, a target population mayconsist of human subjects between the age of 18 and 65, between the ageof 18 and 60, between the age of 18 and 55, between the age of 18 and50, between the age of 18 and 45, between the age of 45 and 65, betweenthe age of 45 and 60, between the age of 18 and 24, between the age of18 and 35, etc. In any one of such target population, the subjects maybe further associated with at least one risk factors, including, withoutlimitation: chronic or genetic disease or disorder andmedication/therapeutics.

In some embodiments, a suitable subject is a non-elderly subject, e.g.,a human individual under the age of 65. In another embodiment, thesubject is below 65, but 18 years or older. Such subjects may be on animmunomodulatory therapy (e.g., a statin therapy, an NSAID therapy,etc.), for example, a statin therapy incorporating at least onesynthetic statin.

As used herein, “enhancing an immune response” may involve, for example,improving or augmenting immunogenicity, as measured by any suitablemethods known or accepted in the art. An “enhanced immune response” isachieved when a statistically significant augmentation of immuneresponses in one measurement or combination of measurements is observedamong a population of subjects meeting certain criteria, i.e., a targetpopulation. A target population may consist of individuals who are on animmunomodulatory therapy (e.g., a statin therapy, an NSAID therapy,etc.). In some embodiments, a subset of such population may represent aparticular category of individuals, such as age groups. The phrase“boosting an immune response” may refer to enhancement of a previouslyprimed immune response in a subject.

The Adjuvant

An “adjuvanted vaccine” comprises one or more adjuvants. Accordingly, insome embodiments, compositions of the invention comprise an adjuvant,which can function to enhance the immune responses (humoral and/orcellular) elicited in a patient who receives the composition. Usefuladjuvants include, but are not limited to: aluminum salt-based adjuvants(e.g., aluminum phosphate and aluminum hydroxide), agonists of Toll-likereceptors (e.g., human TLR1 agonists, human TLR2 agonists, human TLR3agonists, human TLR4 agonists, human TLR5 agonists, human TLR6 agonists,human TLR7 agonists, human TLR8 agonists, human TLR9 agonists, and humanTLR10 agonists), emulsion-based adjuvants, and virus-like particle-basedadjuvants, such as virosomes.

In some embodiments, vaccine adjuvants for use with the inventioncomprise an oil-in-water emulsion.

Oil-in-water emulsions have been found to be particularly suitable foruse in adjuvanting vaccines, including viral vaccines, such as influenzavirus vaccines. Various such emulsions are known, and they typicallyinclude at least one oil and at least one surfactant, with the oil(s)and surfactant(s) being biodegradable (metabolisable) and biocompatible.The oil droplets in the emulsion are generally less than 5 μm indiameter, and ideally the majority of oil droplets in the emulsion havea sub-micron diameter (e.g., at least 90% by number of the oil dropletshave a sub-micron diameter), with these small sizes being achieved witha microfluidiser to provide stable emulsions. Droplets with a size lessthan 220 nm (e.g., less than 220 nm, less than 200 nm, less than 190 nm,less than 180 nm, less than 170 nm, less than 160 nm, less than 150 nm,less than 140 nm, less than 130 nm, less than 120 nm, less than 110 nm,less than 100 nm, etc.) are preferred as they can be subjected to filtersterilization. For example, suitable oil-in-water emulsions includethose with average oil droplet sizes ranging between about 100-200 nm,about 110-200 nm, about 120-200 nm, about 130-200 nm, about 140-200 nm,about 100-190 nm, 100-180 nm, 100-170 nm, 100-160 nm, 100-150 nm,130-190 nm, 135-185 nm, 140-180 nm, 145-175 nm, or 150-170 nm.

The emulsion can comprise oils such as those from an animal (such asfish) or vegetable source. Sources for vegetable oils include nuts,seeds and grains. Peanut oil, soybean oil, coconut oil, and olive oil,the most commonly available, exemplify the nut oils. Jojoba oil can beused, e.g., obtained from the jojoba bean. Seed oils include saffloweroil, cottonseed oil, sunflower seed oil, sesame seed oil and the like.In the grain group, corn oil is the most readily available, but the oilof other cereal grains such as wheat, oats, rye, rice, teff, triticaleand the like may also be used. 6-10 carbon fatty acid esters of glyceroland 1,2-propanediol, while not occurring naturally in seed oils, may beprepared by hydrolysis, separation and esterification of the appropriatematerials starting from the nut and seed oils. Fats and oils frommammalian milk are metabolizable and may therefore be used in thepractice of this invention. The procedures for separation, purification,saponification and other means necessary for obtaining pure oils fromanimal sources are well known in the art. Most fish containmetabolizable oils which may be readily recovered. For example, codliver oil, shark liver oils, and whale oil such as spermaceti exemplifyseveral of the fish oils which may be used herein. A number of branchedchain oils are synthesized biochemically in 5-carbon isoprene units andare generally referred to as terpenoids. Shark liver oil contains abranched, unsaturated terpenoids known as squalene,2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene, which isparticularly preferred herein. Squalane, the saturated analog tosqualene, is also a preferred oil. Fish oils, including squalene andsqualane, are readily available from commercial sources or may beobtained by methods known in the art. Other preferred oils are thetocopherols (see below). Mixtures of oils can be used.

Surfactants can be classified by their ‘HLB’ (hydrophile/lipophilebalance). Preferred surfactants of the invention have a HLB of at least10, preferably at least 15, and more preferably at least 16. Theinvention can be used with surfactants including, but not limited to:the polyoxyethylene sorbitan esters surfactants (commonly referred to asthe Tweens), especially polysorbate 20 and polysorbate 80; copolymers ofethylene oxide (EO), propylene oxide (PO), and/or butylene oxide (BO),sold under the DOWFAX™ tradename, such as linear EO/PO block copolymers;octoxynols, which can vary in the number of repeating ethoxy(oxy-1,2-ethanediyl) groups, with octoxynol-9 (Triton X-100, ort-octylphenoxypolyethoxyethanol) being of particular interest;(octylphenoxy)polyethoxyethanol (IGEPAL CA-630/NP-40); phospholipidssuch as phosphatidylcholine (lecithin); nonylphenol ethoxylates, such asthe Tergitol™ NP series; polyoxyethylene fatty ethers derived fromlauryl, cetyl, stearyl and oleyl alcohols (known as Brij surfactants),such as triethyleneglycol monolauryl ether (Brij 30); and sorbitanesters (commonly known as the SPANs), such as sorbitan trioleate (Span85) and sorbitan monolaurate. Non-ionic surfactants are preferred.Preferred surfactants for including in the emulsion are Tween 80(polyoxyethylene sorbitan monooleate or polysorbate 80), Span 85(sorbitan trioleate), lecithin and Triton X-100.

Mixtures of surfactants can be used, e.g., Tween 80/Span 85 mixtures. Acombination of a polyoxyethylene sorbitan ester such as polyoxyethylenesorbitan monooleate (polysorbate 80) and an octoxynol such ast-octylphenoxypolyethoxyethanol (Triton X-100) is also suitable. Anotheruseful combination comprises laureth 9 plus a polyoxyethylene sorbitanester and/or an octoxynol.

Preferred amounts of surfactants (% by weight) are: polyoxyethylenesorbitan esters (such as polysorbate 80) 0.01 to 1%, in particular about0.1%; octyl- or nonylphenoxy polyoxyethanols (such as Triton X-100, orother detergents in the Triton series) 0.001 to 0.1%, in particular0.005 to 0.02%; polyoxyethylene ethers (such as laureth 9) 0.1 to 20%,preferably 0.1 to 10% and in particular 0.1 to 1% or about 0.5%.

Preferred emulsion adjuvants have an average droplets size of <1 μm,e.g., <750 nm, <500 nm, <400 nm, <300 nm, <250 nm, <220 nm, <200 nm,<190 nm, <180 nm, <170 nm, <160 nm, <150 nm, or smaller. These dropletsizes can conveniently be achieved by techniques such asmicrofluidization and suitable filtration (see, for example,International Patent Publications: WO 2011/067669, WO 2011/067673 and WO2011/067672, the contents of which are incorporated herein).

Provided below are examples of specific oil-in-water emulsion adjuvantsuseful for the invention:

-   -   A submicron emulsion of squalene, polysorbate 80, and sorbitan        trioleate. These three components can be present at a volume        ratio of 10:1:1 or a weight ratio of 39:47:47. The composition        of the emulsion by volume can be about 5% squalene, about 0.5%        polysorbate 80 and about 0.5% sorbitan trioleate. In weight        terms, these ratios become 4.3%>squalene, 0.5%>polysorbate 80        and 0.48%>sorbitan trioleate. This adjuvant is known as “MF59,”        as previously described. The MF59 emulsion may advantageously        include a buffer, such as citrate ions, e.g., 10 mM sodium        citrate buffer.    -   An emulsion of squalene, a tocopherol, and polysorbate 80. The        emulsion may include phosphate buffered saline. It may also        include Span 85 (e.g., at 1%>) and/or lecithin. These emulsions        may have from 2 to 10%>squalene, from 2 to 10%>tocopherol and        from 0.3 to 3%>polysorbate 80, and the weight ratio of        squalene:tocopherol is preferably <1 as this provides a more        stable emulsion. Squalene and polysorbate 80 may be present        volume ratio of about 5:2 or at a weight ratio of about 11:5.        Thus the three components (squalene, tocopherol, polysorbate 80)        may be present at a weight ratio of 1068:1186:485 or around        55:61:25. One such emulsion (“AS03”) can be made by dissolving        Tween 80 in PBS to give a 2%>solution, then mixing 90 ml of this        solution with a mixture of (5 g of DL-a-tocopherol and 5 ml        squalene), then microfluidising the mixture. The resulting        emulsion may have submicron oil droplets e.g., with an average        diameter of between 100 and 250 nm, preferably about 180 nm. The        emulsion may also include a 3-de-O-acylated monophosphoryl lipid        A (3d-MPL). Another useful emulsion of this type may comprise,        per human dose, 0.5-10 mg squalene, 0.5-11 mg tocopherol, and        0.1-4 mg polysorbate 80, e.g., in the ratios discussed above.    -   An emulsion of squalene, a tocopherol, and a Triton detergent        (e.g. Triton X-100). The emulsion may also include a 3d-MPL (see        below). It may contain a buffer, such as a phosphate buffer.    -   An emulsion comprising a polysorbate (e.g., polysorbate 80), a        Triton detergent (e.g., Triton X-100) and a tocopherol (e.g. an        a-tocopherol succinate). The emulsion may include these three        components at a mass ratio of about 75:11:10 (e.g. 750 μg/ml        polysorbate 80, 110 μg/ml Triton X-100 and 100 μg/ml        a-tocopherol succinate), and these concentrations should include        any contribution of these components from antigens. The emulsion        may also include squalene. The emulsion may also include a        3d-MPL (see below). The aqueous phase may contain a buffer, such        as a phosphate buffer.    -   An emulsion of squalane, polysorbate 80 and poloxamer 401        (“Pluronic™ L121”). The emulsion can be formulated in phosphate        buffered saline, pH 7.4. This emulsion is a useful delivery        vehicle for muramyl dipeptides, and has been used with        threonyl-MDP in the “SAF-1” adjuvant (0.05-1% Thr-MDP, 5%        squalane, 2.5% Pluronic L121 and 0.2% polysorbate 80). It can        also be used without the Thr-MDP, as in the “AF” adjuvant (5%        squalane, 1.25% Pluronic L121 and 0.2%>polysorbate 80). Micro        fluidisation is preferred.    -   An emulsion comprising squalene, an aqueous solvent, a        polyoxyethylene alkyl ether hydrophilic nonionic surfactant        (e.g. polyoxyethylene (12) cetostearyl ether) and a hydrophobic        nonionic surfactant (e.g. a sorbitan ester or mannide ester,        such as sorbitan monoleate or ‘Span 80’). The emulsion is        preferably thermoreversible and/or has at least 90% of the oil        droplets (by volume) with a size less than 200 nm. The emulsion        may also include one or more of: alditol; a cryoprotective agent        (e.g. a sugar, such as dodecylmaltoside and/or sucrose); and/or        an alkylpolyglycoside. The emulsion may include a TLR4 agonist.        Such emulsions may be lyophilized.    -   An emulsion of squalene, poloxamer 105 and Abil-Care. The final        concentration (weight) of these components in adjuvanted        vaccines are 5% squalene, 4% poloxamer 105 (pluronic polyol) and        2% Abil-Care 85 (Bis-PEG/PPG-16/16 PEG/PPG-16/16 dimethicone;        caprylic/capric triglyceride).    -   An emulsion having from 0.5-50% of an oil, 0.1-10% of a        phospholipid, and 0.05-5% of a non-ionic surfactant. Preferred        phospholipid components are phosphatidylcholine,        phosphatidylethanolamine, phosphatidylserine,        phosphatidylinositol, phosphatidylglycerol, phosphatidic acid,        sphingomyelin and cardiolipin. Submicron droplet sizes are        advantageous.    -   A submicron oil-in-water emulsion of a non-metabolisable oil        (such as light mineral oil) and at least one surfactant (such as        lecithin, Tween 80 or Span 80). Additives may be included, such        as QuilA saponin, cholesterol, a saponin-lipophile conjugate        (such as GPI-0100, produced by addition of aliphatic amine to        desacylsaponin via the carboxyl group of glucuronic acid),        dimethyidioctadecylammonium bromide and/or        N,N-dioctadecyl-N,N-bis (2-hydroxyethyl)propanediamine.    -   An emulsion in which a saponin (e.g., QuilA and QS21) and a        sterol (e.g., cholesterol) are associated as helical micelles.    -   An emulsion comprising a mineral oil, a non-ionic lipophilic        ethoxylated fatty alcohol, and a non-ionic hydrophilic        surfactant (e.g., an ethoxylated fatty alcohol and/or        polyoxyethylene-polyoxypropylene block copolymer).    -   An emulsion comprising a mineral oil, a non-ionic hydrophilic        ethoxylated fatty alcohol, and a non-ionic lipophilic surfactant        (e.g., an ethoxylated fatty alcohol and/or        polyoxyethylene-polyoxypropylene block copolymer).

In some embodiments an emulsion may be mixed with antigenextemporaneously, at the time of delivery, and thus the adjuvant andantigen may be kept separately in a packaged or distributed vaccine,ready for final formulation at the time of use. In other embodiments anemulsion is mixed with antigen during manufacture, and thus thecomposition is packaged in a liquid adjuvanted form, as in the FLUAD™product. The antigen will generally be in an aqueous form, such that thevaccine is finally prepared by mixing two liquids. The volume ratio ofthe two liquids for mixing can vary (e.g., between 5:1 and 1:5) but isgenerally about 1:1. Where concentrations of components are given in theabove descriptions of specific emulsions, these concentrations aretypically for an undiluted composition, and the concentration aftermixing with an antigen solution will thus decrease.

After the antigen and adjuvant have been mixed, haemagglutinin antigenwill generally remain in aqueous solution but may distribute itselfaround the oil/water interface. In general, little if any haemagglutininwill enter the oil phase of the emulsion.

Where a composition includes a tocopherol, any of the α, β, γ, δ, ε or ξtocopherols can be used (alone or in combination), but α-tocopherols arepreferred. The tocopherol can take several forms, e.g., different saltsand/or isomers. Salts include organic salts, such as succinate, acetate,nicotinate, etc. D-a-tocopherol and DL-a-tocopherol can both be used.Tocopherols are advantageously included in vaccines for use in elderlypatients (e.g., aged 60 years or older or 65 years or older) becausevitamin E has been reported to have a positive effect on the immuneresponse in this patient group. They also have antioxidant propertiesthat may help to stabilize the emulsions. A preferred a-tocopherol isDL-a-tocopherol, and the preferred salt of this tocopherol is thesuccinate. The succinate salt has been found to cooperate withTNF-related ligands in vivo. Moreover, a-tocopherol succinate is knownto be compatible with influenza vaccines and to be a useful preservativeas an alternative to mercurial compounds.

High-Dose Vaccines

In the context of the present disclosure, a high-dose vaccine refers toa vaccine containing an antigen (i.e., immunogen) that is at least abouta twofold the amount of the same antigen contained in a standard dosevaccine, for example, two-fold, three-fold, four-fold, five-fold,six-fold, seven-fold, eight-fold, nine-fold, and ten-fold, which may bealternatively expressed as 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, and 10×,respectively. Standard dose vaccines are those typically commerciallyavailable, which are not specifically labeled or marketed as high-doseor low-dose. In the case of influenza vaccines, a standard dose is about15 μg of antigen per strain (see below for more detail).

The Influenza Virus Antigen

The invention uses an influenza virus antigen, typically comprisinghemagglutinin, to immunize a subject. The antigen will typically beprepared from influenza virions but, as an alternative, antigens such ashaemagglutinin can be expressed in a recombinant host (e.g., in aninsect cell line using a baculovirus vector) and used in purified form.In general, however, antigens may be from virions.

The antigen may take the form of a live virus or, more preferably, aninactivated virus. Chemical means for inactivating a virus includetreatment with an effective amount of one or more of the followingagents: detergents, formaldehyde, formalin, β-propiolactone, or UVlight. Additional chemical means for inactivation include treatment withmethylene blue, psoralen, carboxyfullerene (C60) or a combination of anythereof. Other methods of viral inactivation are known in the art, suchas for example binary ethylamine, acetyl ethyleneimine, or gammairradiation. The INFLEXAL™ product is a whole virion inactivatedvaccine.

Where an inactivated virus is used, the vaccine may comprise wholevirion, split virion, or purified surface antigens (includinghemagglutinin and, usually, also including neuraminidase).

An inactivated but non-whole cell vaccine (e.g., a split virus vaccineor a purified surface antigen vaccine) may include matrix protein, inorder to benefit from the additional T cell epitopes that are locatedwithin this antigen. Thus, a non-whole cell vaccine (particularly asplit vaccine) that includes haemagglutinin and neuraminidase mayadditionally include M1 and/or M2 matrix protein. Nucleoprotein may alsobe present.

Virions can be harvested from virus-containing fluids by variousmethods. For example, a purification process may involve zonalcentrifugation using a linear sucrose gradient solution that includesdetergent to disrupt the virions. Antigens may then be purified, afteroptional dilution, by diafiltration.

Split virions are obtained by treating purified virions with detergentsand/or solvents to produce subvirion preparations, including the‘Tween-ether’ splitting process. Methods of splitting influenza virusesare well known in the art. Splitting of the virus is typically carriedout by disrupting or fragmenting whole virus, whether infectious ornon-infectious with a disrupting concentration of a splitting agent. Thedisruption results in a full or partial solubilisation of the virusproteins, altering the integrity of the virus. Preferred splittingagents are non-ionic and ionic (e.g. cationic) surfactants. Suitablesplitting agents include, but are not limited to: ethyl ether,polysorbate 80, deoxycholate, tri-N-butyl phosphate, alkylglycosides,alkylthioglycosides, acyl sugars, sulphobetaines, betaines,polyoxyethylenealkylethers, N,N-dialkyl-Glucamides, Hecameg,alkylphenoxy-polyethoxyethanols, quaternary ammonium compounds,sarcosyl, CTABs (cetyl trimethyl ammonium bromides), tri-N-butylphosphate, Cetavlon, myristyltrimethylammonium salts, lipofectin,lipofectamine, and DOT-MA, the octyl- or nonylphenoxy polyoxyethanols(e.g. the Triton surfactants, such as Triton X-100 or Triton N101),nonoxynol 9 (NP9) Sympatens-NP/090) polyoxyethylene sorbitan esters (theTween surfactants), polyoxyethylene ethers, polyoxyethlene esters, etc.One useful splitting procedure uses the consecutive effects of sodiumdeoxycholate and formaldehyde, and splitting can take place duringinitial virion purification (e.g. in a sucrose density gradientsolution). Thus a splitting process can involve clarification of thevirion-containing material (to remove non-virion material),concentration of the harvested virions (e.g. using an adsorption method,such as CaHP04 adsorption), separation of whole virions from non-virionmaterial, splitting of virions using a splitting agent in a densitygradient centrifugation step (e.g. using a sucrose gradient thatcontains a splitting agent such as sodium deoxycholate), and thenfiltration (e.g. ultrafiltration) to remove undesired materials. Splitvirions can usefully be resuspended in sodium phosphate-bufferedisotonic sodium chloride solution. The BEGRIVAC™, FLUARIX™, FLUZONE™ andFLUSHIELD™ products are split vaccines.

Purified surface antigen vaccines comprise the influenza surfaceantigens haemagglutinin and, typically, also neuraminidase. Processesfor preparing these proteins in purified form are well known in the art.The FLUVIRIN™, AGRIPPAL™ and INFLUVAC™ products are subunit vaccines.

Another form of inactivated influenza antigen is the virosome (nucleicacid free viral-like liposomal particles). Virosomes can be prepared bysolubilization of influenza virus with a detergent followed by removalof the nucleocapsid and reconstitution of the membrane containing theviral glycoproteins. An alternative method for preparing virosomesinvolves adding viral membrane glycoproteins to excess amounts ofphospholipids, to give liposomes with viral proteins in their membrane.The invention can be used to store bulk virosomes, as in the INFLEXAL V™product. In some embodiments, the influenza antigen is not in the formof a virosome.

The influenza virus may be attenuated. The influenza virus may betemperature-sensitive. The influenza virus may be cold-adapted. Thesethree features are particularly useful when using live virus as avaccine antigen.

With respect to inactivated influenza vaccines currently available, HAis the main immunogen, and vaccine doses are standardized by referenceto HA levels, typically measured by SRID. Existing vaccines typicallycontain about 15 μg of HA per strain, although lower doses can be used,e.g., for children, or in pandemic situations, or when used inconjunction with an adjuvant. Fractional doses such as ½ (e.g., 7.5 μgHA per strain), ¼ and ⅛ have been used, as have higher doses (e.g., 2×,3× or 9× doses).

Thus, vaccines of the present invention may include between about 0.1and about 150 μg of an antigen. For example, HA per influenza strain,preferably between 0.1 and 50 μg, e.g., 0.1-20 μg, 0.1-15 μg, 0.1-10 μg,0.1-7 μg, 0.5-5 μg, etc. Particular doses include, e.g., about 45 μg,about 30 μg, about 15 μg, about 10 μg, about 7.5 μg, about 5 μg, about3.8 μg, about 1.9 μg, about 1.5 μg, etc. per strain.

For live vaccines, dosing is measured by median tissue cultureinfectious dose (TCID50) rather than HA content, and a TCID50 of between10⁶ and 10⁸ (preferably between 10^(6.5)-10^(7.5)) per strain istypical.

As used herein, a “high-dose influenza vaccine” may contain betweenabout 30 μg and 150 μg of an antigen (e.g., HA) per strain, as opposedto the standard-dose vaccine, which typically contains 15 μg of anantigen (e.g., HA) per strain. Thus, in some embodiments, a high-dosevaccine contains about 30 μg, about 35 μg, about 40 μg, about 45 μg,about 50 μg, about 55 μg, about 60 μg, about 65 μg, about 70 μg, about75 μg, about 80 μg, about 85 μg, about 90 μg about 100 μg about 110 μgabout 120 μg about 130 μg about 140 μg about 150 μg of antigen perstrain.

A “low-dose influenza vaccine,” on the other hand, refers to a vaccinecomposition that contains less than 15 μg of an antigen (e.g., HA) perstrain, for example, about 12.5 μg, about 10 μg, about 7.5 μg, about 5μg, about 4 μg, about 3 μg, about 2.5 μg. In some embodiments, any ofhigh-dose vaccines, standard-dose vaccines, and low-dose vaccines may beadjuvanted.

Influenza virus strains for use in vaccines change from season toseason. In the current inter-pandemic period, vaccines typically includetwo influenza A strains (H1N1 and H3N2) and one or two influenza Bstrain, and trivalent or tetravalent vaccines are typical for use withthe invention. Compositions of the invention comprise antigen frominfluenza B virus and optionally comprise antigen from at least oneinfluenza A virus. Where the composition of the invention comprisesantigen from influenza A virus(es), the invention may use seasonaland/or pandemic strains. Depending on the season and on the nature ofthe antigen included in the vaccine, the invention may include (andprotect against) one or more of influenza A virus hemagglutinin subtypesH1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 or H16.The vaccine may additionally include neuraminidase from any of NAsubtypes N1, N2, N3, N4, N5, N6, N7, N8 or N9.

The invention can thus be used with pandemic influenza A virus strains.Characteristics of a pandemic strain are: (a) it contains a newhemagglutinin compared to the hemagglutinins in currently-circulatinghuman strains, i.e., one that has not been evident in the humanpopulation for over a decade (e.g., H2), or has not previously been seenat all in the human population (e.g., H5, H6 or H9, that have generallybeen found only in bird populations), such that the vaccine recipientand the general human population are immunologically naive to thestrain's hemagglutinin; (b) it is capable of being transmittedhorizontally in the human population; and (c) it is pathogenic tohumans. Pandemic strains include, but are not limited to, H2, H5, H7 orH9 subtype strains, e.g., H5N1, H5N3, H9N2, H2N2, H7N1 and H7N7 strains.Within the H5 subtype, a virus may fall into a number of clades e.g.clade 1 or clade 2. Six sub-clades of clade 2 have been identified withsub-clades 1, 2 and 3 having a distinct geographic distribution and areparticularly relevant due to their implication in human infections.

Influenza B virus currently does not display different HA subtypes, butinfluenza B virus strains do fall into two distinct lineages. Theselineages emerged in the late 1980s and have HAs which can beantigenically and/or genetically distinguished from each other. Currentinfluenza B virus strains are either B/Victoria/2/87-like orB/Yamagata/16/88-like. These strains are usually distinguishedantigenically, but differences in amino acid sequences have also beendescribed for distinguishing the two lineages, e.g.,B/Yamagata/16/88-like strains often (but not always) have HA proteinswith deletions at amino acid residue 164, numbered relative to the‘Lee40’ HA sequence. The invention can be used with antigens from a Bvirus of either lineage, or with antigens from both lineages.

Where a vaccine includes more than one strain of influenza, thedifferent strains are typically grown separately and are mixed after theviruses have been harvested and antigens have been prepared. Thus amanufacturing process of the invention may include the step of mixingantigens from more than one influenza strain.

An influenza virus used with the invention may be a reassortant strain,and may have been obtained by reverse genetics techniques. Reversegenetics techniques allow influenza viruses with desired genome segmentsto be prepared in vitro using plasmids. Typically, it involvesexpressing (a) DNA molecules that encode desired viral RNA moleculese.g. from poll promoters or bacteriophage RNA polymerase promoters, and(b) DNA molecules that encode viral proteins, e.g., from poIIIpromoters, such that expression of both types of DNA in a cell leads toassembly of a complete intact infectious virion. The DNA preferablyprovides all of the viral RNA and proteins, but it is also possible touse a helper virus to provide some of the RNA and proteins.Plasmid-based methods using separate plasmids for producing each viralRNA can be used, and these methods will also involve the use of plasmidsto express all or some (e.g., just the PB 1, PB2, PA and NP proteins) ofthe viral proteins, with up to 12 plasmids being used in some methods.To reduce the number of plasmids needed, a recent approach combines aplurality of RNA polymerase I transcription cassettes (for viral RNAsynthesis) on the same plasmid (e.g., sequences encoding 1, 2, 3, 4, 5,6, 7 or all 8 influenza A vRNA segments), and a plurality ofprotein-coding regions with RNA polymerase II promoters on anotherplasmid (e.g., sequences encoding 1, 2, 3, 4, 5, 6, 7 or all 8 influenzaA mRNA transcripts). In some embodiments of the invention, methodsinvolve: (a) PB 1, PB2 and PA mRNA-encoding regions on a single plasmid;and (b) all 8 vRNA-encoding segments on a single plasmid. Including theNA and HA segments on one plasmid and the six other segments on anotherplasmid can also facilitate matters.

As an alternative to using poll promoters to encode the viral RNAsegments, it is possible to use bacteriophage polymerase promoters. Forinstance, promoters for the SP6, T3 or T7 polymerases can convenientlybe used. Because of the species-specificity of poll promoters,bacteriophage polymerase promoters can be more convenient for many celltypes (e.g. MDCK), although a cell must also be transfected with aplasmid encoding the exogenous polymerase enzyme.

In other techniques it is possible to use dual poII and poIII promotersto simultaneously code for the viral RNAs and for expressible mRNAs froma single template.

Thus an influenza A virus may include one or more RNA segments from anA/PR/8/34 virus (typically 6 segments from A/PR/8/34, with the HA and Nsegments being from a vaccine strain, i.e. a 6:2 reassortant). It mayalso include one or more RNA segments from an A/WSN/33 virus, or fromany other virus strain useful for generating reassortant viruses forvaccine preparation. An influenza A virus may include fewer than 6(e.g., 0, 1, 2, 3, 4 or 5) viral segments from an AA/6/60 influenzavirus (A/Ann Arbor/6/60). An influenza B virus may include fewer than 6(e.g., 0, 1, 2, 3, 4 or 5) viral segments from an AA/1/66 influenzavirus (B/Ann Arbor/1/66). Typically, the invention protects against astrain that is capable of human-to-human transmission, and so thestrain's genome will usually include at least one RNA segment thatoriginated in a mammalian (e.g., in a human) influenza virus. It mayinclude NS segment that originated in an avian influenza virus.

Strains whose antigens can be included in the compositions may beresistant to antiviral therapy (e.g., resistant to oseltamivir and/orzanamivir), including resistant pandemic strains.

HA used with the invention may be a natural HA as found in a virus, ormay have been modified. For instance, it is known to modify HA to removedeterminants (e.g., hyper-basic regions around the cleavage site betweenHA1 and HA2) that cause a virus to be highly pathogenic in avianspecies, as these determinants can otherwise prevent a virus from beinggrown in eggs.

The viruses used as the source of the antigens can be grown either oneggs (e.g., specific pathogen free eggs) or on cell culture. The currentstandard method for influenza virus growth uses embryonated hen eggs,with virus being purified from the egg contents (allantoic fluid). Morerecently, however, viruses have been grown in animal cell culture and,for reasons of speed and patient allergies, this growth method ispreferred.

The cell line will typically be of mammalian origin. Suitable mammaliancells of origin include, but are not limited to, hamster, cattle,primate (including humans and monkeys) and dog cells, although the useof primate cells is not preferred. Various cell types may be used, suchas kidney cells, fibroblasts, retinal cells, lung cells, etc. Examplesof suitable hamster cells are the cell lines having the names BHK21 orHKCC. Suitable monkey cells are e.g. African green monkey cells, such askidney cells as in the Vero cell line. Suitable dog cells are, e.g.,kidney cells, as in the CLDK and MDCK cell lines.

Thus suitable cell lines include, but are not limited to: MDCK; CHO;CLDK; HKCC; 293T; BHK; Vero; MRC-5; PER.C6; FRhL2; WI-38; etc. Suitablecell lines are widely available, e.g., from the American Type CellCulture (ATCC) collection, from the Coriell Cell Repositories, or fromthe European Collection of Cell Cultures (ECACC). For example, the ATCCsupplies various different Vero cells under catalog numbers CCL-81,CCL-81.2, CRL-1586 and CRL-1587, and it supplies MDCK cells undercatalog number CCL-34. PER.C6 is available from the ECACC under depositnumber 96022940.

The most preferred cell lines are those with mammalian-typeglycosylation. As a less-preferred alternative to mammalian cell lines,virus can be grown on avian cell lines, including cell lines derivedfrom ducks (e.g. duck retina) or hens. Examples of avian cell linesinclude avian embryonic stem cells and duck retina cells. Suitable avianembryonic stem cells include the EBx cell line derived from chickenembryonic stem cells, EB45, EB14, and EB 14-074. Chicken embryofibroblasts (CEF) may also be used. Rather than using avian cells,however, the use of mammalian cells means that vaccines can be free fromavian DNA and egg proteins (such as ovalbumin and ovomucoid), therebyreducing allergenicity.

The most preferred cell lines for growing influenza viruses are MDCKcell lines, derived from Madin Darby canine kidney. The original MDCKcell line is available from the ATCC as CCL-34, but derivatives of thiscell line may also be used. For instance, a MDCK cell line has beenadapted for growth in suspension culture (‘MDCK 33016’, deposited as DSMACC 2219). Similarly, a MDCK-derived cell line has been developed whichgrows in suspension in serum-free culture (B-702′, deposited as FERMBP-7449). Non-tumorigenic MDCK cells have been described, including‘MDCK-S’ (ATCC PTA-6500), ‘MDCK-SF101’ (ATCC PTA-6501), ‘MDCK-SF102’(ATCC PTA-6502) and ‘MDCK-SF103’ (PTA-6503). MDCK cell lines with highsusceptibility to infection have been developed, including‘MDCKMDCK.5F1’ cells (ATCC CRL-12042). Any of these MDCK cell lines canbe used.

Virus may be grown on cells in adherent culture or in suspension.Microcarrier cultures can also be used. In some embodiments, the cellsmay thus be adapted for growth in suspension.

Cell lines are preferably grown in serum-free culture media and/orprotein free media. A medium is referred to as a serum-free medium inthe context of the present invention in which there are no additivesfrom serum of human or animal origin. The cells growing in such culturesnaturally contain proteins themselves, but a protein-free medium isunderstood to mean one in which multiplication of the cells occurs withexclusion of proteins, growth factors, other protein additives andnon-serum proteins, but can optionally include proteins such as trypsinor other proteases that may be necessary for viral growth.

Cell lines supporting influenza virus replication are preferably grownbelow 37° C. (e.g., 30-36° C., or at about 30° C., 31° C., 32° C., 33°C., 34° C., 35° C., 36° C.) during viral replication.

Methods for propagating influenza virus in cultured cells generallyincludes the steps of inoculating a culture of cells with an inoculum ofthe strain to be grown, cultivating the infected cells for a desiredtime period for virus propagation, such as for example as determined byvirus titer or antigen expression (e.g., between 24 and 168 hours afterinoculation) and collecting the propagated virus. The cultured cells areinoculated with a virus (measured by PFU or TCID50) to cell ratio of1:500 to 1:1, preferably 1:100 to 1:5, more preferably 1:50 to 1:10. Thevirus is added to a suspension of the cells or is applied to a monolayerof the cells, and the virus is absorbed on the cells for at least 60minutes but usually less than 300 minutes, preferably between 90 and 240minutes at 25° C. to 40° C., preferably 28° C. to 37° C. The infectedcell culture (e.g., monolayers) may be removed either by freeze-thawingor by enzymatic action to increase the viral content of the harvestedculture supernatants. The harvested fluids are then either inactivatedor stored frozen. Cultured cells may be infected at a multiplicity ofinfection (“m.o.i.”) of about 0.0001 to 10, preferably 0.002 to 5, morepreferably to 0.001 to 2. Still more preferably, the cells are infectedat an m.o.i of about 0.01. Infected cells may be harvested 30 to 60hours post infection. Preferably, the cells are harvested 34 to 48 hourspost infection. Still more preferably, the cells are harvested 38 to 40hours post infection. Proteases (typically trypsin) are generally addedduring cell culture to allow viral release, and the proteases can beadded at any suitable stage during the culture e.g. before inoculation,at the same time as inoculation, or after inoculation.

In preferred embodiments, particularly with MDCK cells, a cell line isnot passaged from the master working cell bank beyond 40population-doubling levels.

The viral inoculum and the viral culture are preferably free from (i.e.,will have been tested for and given a negative result for contaminationby) herpes simplex virus, respiratory syncytial virus, parainfluenzavirus 3, SARS coronavirus, adenovirus, rhinovirus, reoviruses,polyomaviruses, birnaviruses, circoviruses, and/or parvoviruses. Absenceof herpes simplex viruses is particularly preferred.

Where virus has been grown on a cell line then it is standard practiceto minimize the amount of residual cell line DNA in the final vaccine,in order to minimize any oncogenic activity of the DNA.

Thus a vaccine composition prepared according to the inventionpreferably contains less than 10 ng (preferably less than 1 ng, and morepreferably less than 100 pg) of residual host cell DNA per dose,although trace amounts of host cell DNA may be present.

Vaccines containing <10 ng (e.g. <1 ng, <100 pg) host cell DNA per 15 μgof haemagglutinin are preferred, as are vaccines containing <10 ng (e.g.<1 ng, <100 pg) host cell DNA per 0.25 ml volume.

Vaccines containing <10 ng (e.g. <1 ng, <100 pg) host cell DNA per 5 μgof haemagglutinin are more preferred, as are vaccines containing <10 ng(e.g. <1 ng, <100 pg) host cell DNA per 0.5 ml volume.

It is preferred that the average length of any residual host cell DNA isless than 500 bp, e.g., less than 400 bp, less than 300 bp, less than200 bp, less than 100 bp, etc.

Contaminating DNA can be removed during vaccine preparation usingstandard purification procedures e.g. chromatography, etc. Removal ofresidual host cell DNA can be enhanced by nuclease treatment e.g. byusing a DNase. A convenient method for reducing host cell DNAcontamination has been described in the literature, involving a two-steptreatment, first using a DNase (e.g. Benzonase), which may be usedduring viral growth, and then a cationic detergent (e.g. CTAB), whichmay be used during virion disruption. Removal by 3-propiolactonetreatment can also be used.

Measurement of residual host cell DNA is now a routine regulatoryrequirement for biologicals and is within the normal capabilities of theskilled person. The assay used to measure DNA will typically be avalidated assay. The performance characteristics of a validated assaycan be described in mathematical and quantifiable terms, and itspossible sources of error will have been identified. The assay willgenerally have been tested for characteristics such as accuracy,precision, specificity. Once an assay has been calibrated (e.g. againstknown standard quantities of host cell DNA) and tested then quantitativeDNA measurements can be routinely performed. Three main techniques forDNA quantification can be used: hybridization methods, such as Southernblots or slot blots; immunoassay methods, such as the Threshold™ System;and quantitative PCR. These methods are all familiar to the skilledperson, although the precise characteristics of each method may dependon the host cell in question e.g. the choice of probes forhybridization, the choice of primers and/or probes for amplification,etc. The Threshold™ system from Molecular Devices is a quantitativeassay for picogram levels of total DNA, and has been used for monitoringlevels of contaminating DNA in biopharmaceuticals. A typical assayinvolves non-sequence-specific formation of a reaction complex between abiotinylated ssDNA binding protein, a urease-conjugated anti-ssDNAantibody, and DNA. All assay components are included in the completeTotal DNA Assay Kit available from the manufacturer. Various commercialmanufacturers offer quantitative PCR assays for detecting residual hostcell DNA, e.g., AppTec™ Laboratory Services, BioReliance™, AltheaTechnologies, etc. A comparison of a chemiluminescent hybridisationassay and the total DNA Threshold™ system for measuring host cell DNAcontamination of a human viral vaccine has been described.

Pharmaceutical Compositions

Compositions of the invention are pharmaceutically acceptable.Compositions of the invention include vaccines. They may includecomponents in addition to the antigen and adjuvant, e.g., they willtypically include one or more pharmaceutical carrier(s) and/orexcipient(s), which are well known in the art.

The composition may include preservatives such as thiomersal or2-phenoxyethanol. It is preferred, however, that the vaccine should besubstantially free from (e.g., less than 5 μg/ml) mercurial material,e.g., thiomersal-free. Vaccines containing no mercury are morepreferred, and a-tocopherol succinate can be included as an alternativeto mercurial compounds. Preservative-free vaccines are most preferred.

To control tonicity, it is preferred to include a physiological salt,such as a sodium salt. Sodium chloride (NaCl) is preferred, which may bepresent at between 1 and 20 mg/ml. Other salts that may be presentinclude potassium chloride, potassium dihydrogen phosphate, disodiumphosphate dehydrate, magnesium chloride, calcium chloride, etc.

Compositions will generally have an osmolality of between 200 mOsm/kgand 400 mOsm/kg, preferably between 240-360 mOsm/kg, and will morepreferably fall within the range of 290-310 mOsm/kg. Osmolality haspreviously been reported not to have an impact on pain caused byvaccination, but keeping osmolality in this range is neverthelesspreferred.

Compositions may include one or more buffers. Typical buffers include: aphosphate buffer; a Tris buffer; a borate buffer; a succinate buffer; ahistidine buffer (particularly with an aluminum hydroxide adjuvant); ora citrate buffer. Buffers will typically be included in the 5-20 mMrange.

The pH of a composition will generally be between 5.0 and 8.1, and moretypically between 6.0 and 8.0, e.g., 6.5 and 7.5, or between 7.0 and7.8. A manufacturing process of the invention may therefore include astep of adjusting the pH of the bulk vaccine prior to packaging.

The composition is preferably sterile. The composition is preferablynon-pyrogenic, e.g., containing <1 EU (endotoxin unit, a standardmeasure) per dose, and preferably <0.1 EU per dose. The composition ispreferably gluten free.

Compositions of the invention may include detergent e.g., apolyoxyethylene sorbitan ester surfactant (known as “Tweens”), anoctoxynol (such as octoxynol-9 (Triton X-100) ort-octylphenoxypolyethoxyethanol), a cetyl trimethyl ammonium bromide(“CTAB”), or sodium deoxycholate, particularly for a split or surfaceantigen vaccine. The detergent may be present only at trace amounts.Thus the vaccine may include less than 1 mg/ml each of octoxynol-10 andpolysorbate 80. Other residual components in trace amounts could beantibiotics (e.g., neomycin, kanamycin, polymyxin B).

The composition may include material for a single immunization, or mayinclude material for multiple immunizations, which is also referred toas a “multidose” kit. The inclusion of a preservative is preferred inmultidose arrangements. As an alternative (or in addition) to includinga preservative in multidose compositions, the compositions may becontained in a container having an aseptic adaptor for removal ofmaterial.

Influenza vaccines are typically administered in a dosage volume (unitdose) of about 0.5 ml, although a half dose (i.e., about 0.25 ml) may beadministered to children according to the invention.

Compositions and kits are preferably stored at between 2° C. and 8° C.They should not be frozen. They should ideally be kept out of directlight.

The antigen and emulsion in a composition will typically be inadmixture, although they may initially be presented in the form of a kitof separate components for extemporaneous admixing. Compositions willgenerally be in aqueous form when administered to a subject.

Kits of the Invention

Compositions of the invention may be prepared extemporaneously, at thetime of delivery. Thus the invention provides kits including the variouscomponents ready for mixing. The kit allows the adjuvant and the antigento be kept separately until the time of use.

The components are physically separate from each other within the kit,and this separation can be achieved in various ways. For instance, thetwo components may be in two separate containers, such as vials. Thecontents of the two vials can then be mixed e.g. by removing thecontents of one vial and adding them to the other vial, or by separatelyremoving the contents of both vials and mixing them in a thirdcontainer.

In a preferred arrangement, one of the kit components is in a syringeand the other is in a container such as a vial. The syringe can be used(e.g., with a needle) to insert its contents into the second containerfor mixing, and the mixture can then be withdrawn into the syringe. Themixed contents of the syringe can then be administered to a patient,typically through a new sterile needle. Packing one component in asyringe eliminates the need for using a separate syringe for patientadministration.

In another preferred arrangement, the two kit components are heldtogether but separately in the same syringe, e.g., a dual-chambersyringe. When such syringe is actuated (e.g., during administration to apatient) then the contents of the two chambers are mixed. Thisarrangement avoids the need for a separate mixing step at the time ofuse.

The kit components will generally be in aqueous form. In somearrangements, a component (typically an antigen component rather than anadjuvant component) is in dry form (e.g., in a lyophilized form), withthe other component being in aqueous form. The two components can bemixed in order to reactivate the dry component and give an aqueouscomposition for administration to a patient. A lyophilized componentwill typically be located within a vial rather than a syringe. Driedcomponents may include stabilizers such as lactose, sucrose or mannitol,as well as mixtures thereof e.g. lactose/sucrose mixtures,sucrose/mannitol mixtures, etc. One possible arrangement uses an aqueousadjuvant component in a pre-filled syringe and a lyophilized antigencomponent in a vial.

Packaging of Compositions or Kit Components

Suitable containers for compositions of the invention (or kitcomponents) include vials, syringes (e.g. disposable syringes), nasalsprays, etc. These containers should be sterile.

Where a composition/component is located in a container, such as a vial,such container is optionally made of a glass or plastic material. Insome embodiments, such container is a siliconized container. The vial ispreferably sterilized before the composition is added to it. To avoidproblems with latex-sensitive patients, vials are preferably sealed witha latex-free stopper, and the absence of latex in all packaging materialis preferred. The vial may include a single dose of vaccine, or it mayinclude more than one dose (i.e., a “multidose” vial), e.g., 10 doses.In some embodiments, vials are made of colorless glass.

A vial can have a cap (e.g., a Luer lock) adapted such that a pre-filledsyringe can be inserted into the cap, the contents of the syringe can beexpelled into the vial (e.g., to reconstitute lyophilized materialtherein), and the contents of the vial can be removed back into thesyringe. After removal of the syringe from the vial, a needle can thenbe attached and the composition can be administered to a patient. Thecap is preferably located inside a seal or cover, such that the seal orcover has to be removed before the cap can be accessed. A vial may havea cap that permits aseptic removal of its contents, particularly formultidose vials.

Where a component is packaged into a syringe, the syringe may have aneedle attached to it. If a needle is not attached, a separate needlemay be supplied with the syringe for assembly and use. Such a needle maybe sheathed. Safety needles are preferred. 1-inch 23-gauge, 1-inch25-gauge and ⅝-inch 25-gauge needles are typical. Syringes may beprovided with peel-off labels on which the lot number, influenza seasonand expiration date of the contents may be printed, to facilitate recordkeeping. The plunger in the syringe preferably has a stopper to preventthe plunger from being accidentally removed during aspiration. Thesyringes may have a latex rubber cap and/or plunger. Disposable syringescontain a single dose of vaccine. The syringe will generally have a tipcap to seal the tip prior to attachment of a needle, and the tip cap ispreferably made of a butyl rubber. If the syringe and needle arepackaged separately then the needle is preferably fitted with a butylrubber shield. Useful syringes are those marketed under the trade name“Tip-Lok”™.

Containers may be marked to show a half-dose volume e.g. to facilitatedelivery to children. For instance, a syringe containing a 0.5 ml dosemay have a mark showing a 0.25 ml volume.

Where a glass container (e.g., a syringe or a vial) is used, then it ispreferred to use a container made from a borosilicate glass rather thanfrom a soda lime glass. In some embodiments, a container is asiliconized container, such as a siliconized glass container.

A kit or composition may be packaged (e.g., in the same box) with aleaflet including details of the vaccine e.g. instructions foradministration, details of the antigens within the vaccine, etc. Theinstructions may also contain warnings e.g. to keep a solution ofadrenaline readily available in case of anaphylactic reaction followingvaccination, etc.

Methods of Treatment, and Administration of the Vaccine

Compositions of the invention are suitable for administration to humanpatients, and the invention provides a method of raising an immuneresponse in a patient, comprising the step of administering acomposition of the invention to the patient. As described above, in someembodiments, the patient is an immunocompromised individual on animmunomodulatory therapy (e.g., a statin therapy, an NSAID therapy,etc.).

These methods and uses will generally be used to generate an antibodyresponse, preferably a protective antibody response. Methods forassessing antibody responses, neutralising capability and protectionafter influenza virus vaccination are well known in the art. Humanstudies have shown that antibody titers against hemagglutinin of humaninfluenza virus are correlated with protection (a serum samplehemagglutination-inhibition titer of about 30-40 gives around 50%protection from infection by a homologous virus). Antibody responses aretypically measured by hemagglutination inhibition (HI), bymicroneutralization (Micro-NT), and/or by single radial hemolysis (SRH)but any other suitable methods may be employed. Such assay techniquesare well known in the art.

As used herein, the terms “administering,” “administration” and thelike, refer to making a pharmaceutical composition (such as vaccines)available to a subject's body, locally or systemically, by any suitableroute(s). Compositions of the invention can be administered in variousways. The most preferred immunization route is by intramuscularinjection (e.g., into the arm or leg), but other available routesinclude subcutaneous injection, intranasal, oral, mucosal, transmucosal,parenteral, intradermal, transcutaneous, transdermal, etc. A morecomplete list of administration routes can be found at:www.fda.gov/Drugs/DevelopmentApprovalProcess/FormsSubmissionRequirements/ElectronicSubmissions/DataStandardsManualmonographs/ucm071667.htm.

Preferred compositions of the invention will satisfy 1, 2 or 3 of theCHMP criteria for adult efficacy for each influenza strain, even thoughthey are administered to children. These criteria are: (1) >70%seroprotection; (2) >40% seroconversion or significant increase; and/or(3) a GMT increase of >2.5-fold. In elderly (>65 years), these criteriaare: (1) >60% seroprotection; (2) >30% seroconversion; and/or (3) a GMTincrease of >2-fold.

The invention is particularly useful for raising immune responses thatare protective against different influenza virus strains, such as A andB virus strains.

As used herein, the terms “effective amount” and “effective dose” referto an amount or dose of a compound or composition that is sufficient tofulfill its intended purpose(s), i.e., eliciting a desired biological ormedicinal response in a tissue or subject at an acceptable benefit/riskratio. For example, in certain embodiments of the present invention, thepurpose(s) may be to induce or augment a desired immune response in asubject. The relevant intended purpose may be objective (i.e.,measurable by some test or marker) or subjective (i.e., subject gives anindication of or feels an effect). In some embodiments, an effectiveamount is an amount that, when administered to a population of subjectsthat meets certain criteria (for example, as determined by medical ortreatment history, genetic or age profile, etc.), a statisticallysignificant response is obtained among the population. An effectiveamount is commonly administered in a dosing regimen that may comprisemultiple unit doses. For any particular pharmaceutical agent, aneffective amount (and/or an appropriate unit dose within an effectivedosing regimen) may vary, for example, depending on route ofadministration, population profiles, and so on. Those of ordinary skillin the art will appreciate that in some embodiments of the invention, aunit dosage may be considered to contain an effective amount if itcontains an amount appropriate for administration in the context of adosage regimen correlated with a positive outcome.

Treatment with compositions of the invention can be by a single doseschedule or a multiple dose schedule. Thus, in any particular influenzaseason (e.g., in a given 12 month period, typically in autumn or winter)a patient may receive a single dose of a composition of the invention ormore than one dose of composition of the invention (e.g., two doses).Where treatment comprises administration of two or more doses ofcompositions of the invention, each dose will generally not be given atsubstantially the same time i.e., they will not be administered duringthe same visit to a vaccination center. The time between successiveadministration of compositions of the invention is typically at least ndays, where n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 42, 49, 56 or more. Typically, two doses areadministered at least 1 week apart (e.g., about 2 weeks, about 3 weeks,about 4 weeks, about 6 weeks, about 8 weeks, about 12 weeks, about 16weeks, etc.). Giving two doses separated by from 25-30 days (e.g., 28days) is particularly useful. The time between doses will typically beno longer than 6 months. The doses may be given about 4 weeks apart fromeach other e.g., at day 0 and then at about day 28. Separation of dosingin this way has been found to give good immune responses.

Where compositions of the invention are used in a primary immunizationschedule, dose(s) with compositions of the invention are followed byadministration of one or more booster vaccines (e.g., 1, 2, 3, or morebooster vaccines). Suitable timing between priming and administration ofbooster vaccine can be routinely determined. The time betweenadministration of a priming dose and administration of a booster vaccineis typically at least p months, where p is selected from 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, or more. Ideally, p is 9 or more,and may be within the range of 9-30.

In a multiple dose schedule the various doses may be given by the sameor different routes, e.g., a parenteral prime and mucosal boost, amucosal prime and parenteral boost, etc. Typically, but not necessarily,they will be given by the same route. Vaccines produced by the inventionmay be administered to patients at substantially the same time as (e.g.,during the same medical consultation or visit to a healthcareprofessional or vaccination center) other vaccines, e.g., atsubstantially the same time as a measles vaccine, a mumps vaccine, arubella vaccine, a MMR vaccine, a varicella vaccine, a MMRV vaccine, adiphtheria vaccine, a tetanus vaccine, a pertussis vaccine, a DTPvaccine, a conjugated H. influenzae type b vaccine, an inactivatedpoliovirus vaccine, a hepatitis B virus vaccine, a meningococcalconjugate vaccine (such as a tetravalent A-C-W135-Y vaccine), apneumococcal conjugate vaccine, etc.

Similarly, vaccines of the invention may be administered to patients atsubstantially the same time as (e.g., during the same medicalconsultation or visit to a healthcare professional) an antiviralcompound, and in particular an antiviral compound active againstinfluenza virus (e.g., oseltamivir and/or zanamivir). These antiviralsinclude neuraminidase inhibitors, such as a(3R,4R,5S)-4-acetylamino-5-amino-3(1-ethylpropoxy)-1-cyclohexene-1-carboxylicacid or5-(acetylamino)-4-[(aminoiminomethyl)-amino]-2,6-anhydro-3,4,5-trideoxy-D-glycero-D-galactonon-2-enonicacid, including esters thereof (e.g. the ethyl esters) and salts thereof(e.g. the phosphate salts). A preferred antiviral is(3R,4R,5S)-4-acetylamino-5-amino-3(1-ethylpropoxy)-1-cyclohexene-1-carboxylicacid, ethyl ester, phosphate (1:1), also known as oseltamivir phosphate(TAMIFLU™).

High-Dose Flu Vaccines for Statin Recipients

For embodiments in which a high-dose vaccine is administered to asubject of a target population, such vaccine may be an unadjuvantedvaccine. In some embodiments, a target population of the presentinvention refers to human subjects on an immunomodulatory therapy (e.g.,a statin therapy, an NSAID therapy, etc.), regardless of the subjects'age, while in other embodiments, a target population of the presentinvention refers to human subjects on an immunomodulatory therapy (e.g.,a statin therapy, an NSAID therapy, etc.) who are also of a particularage group, e.g., aged 65 or older; aged between 18 and 64, etc. Suchsubjects may be on a statin therapy comprising a non-synthetic statin, asynthetic statin, or combination thereof. Although any of suchpopulations will benefit from the invention described herein, it may beparticularly beneficial to those who are on a synthetic statin therapy,who are on a long-term statin therapy, or both.

High-dose vaccines include monovalent and multivalent (e.g., trivalentand tetravalent) influenza vaccines and may contain a high-dose (e.g.,50 μg, 60 μg, 70 μg, 80 μg, 90 μg) each of the strains. Such vaccinecompositions may be formulated as an injectable sterile suspension(e.g., 0.5 mL) containing suitable antigen(s). Virus used to producesuch compositions may optionally be produced in embryonated chickeneggs, may optionally be inactivated (e.g., with formaldehyde), mayoptionally be split (e.g., with a nonionic detergent), and may comprisean A/(H1N1)-like strain, an A/(H3N2)-like strain, and a B strain. Togive but a few examples, A/California/7/2009 (H1N1), A/Victoria/210/2009(H3N2), and B/Brisbane/60/2008 strains may be formulated for one season,while A/California/7/2009 (H1N1), A/Victoria/361/2011 (H3N2), andB/Texas/6/2011 (B/Wisconsin/1/2010-like virus) strains may be formulatedfor another season. Such vaccine compositions may optionally be providedin ready-to-use syringes (such as 0.5 mL) and may be administeredintramuscularly (IM) to a patient on a statin therapy, optionally in thedeltoid area.

General

Throughout the specification, including the claims, where the contextpermits, the term “comprising” and variants thereof, such as “comprises”or “comprising,” are to be interpreted as including the stated element(e.g., integer) or elements (e.g., integers) without necessarilyexcluding any other elements (e.g., integers).

The term “substantially” does not exclude “completely” e.g., acomposition which is “substantially free” from Y may be completely freefrom Y. Where necessary, the word “substantially” may be omitted fromthe definition of the invention.

The term “about” in relation to a numerical value n refers to a range ofnumerical values considered typical or acceptable, e.g., withinstatistical errors, deviations or variations within a particularpopulation, from which a relevant set of data is obtained for certainmeasurements or analyses. Those skilled in the art can readilyunderstand such ranges. For example, a numerical value n may mean(n±1%), (n±1%), (n±2%), (n±3%), (n±4%), (n±5%), (n±6%), (n±7%), (n±8%),(n±9%), (n±10%), (n±1%), (n±12%), (n±13%), (n±14%), (n±15%), (n±16%),(n±17), (n±18%), (n±19%), (n±20%) and so on.

Unless specifically stated, a process comprising a step of mixing two ormore components does not require any specific order of mixing. Thuscomponents can be mixed in any order. Where there are three componentsthen two components can be combined with each other, and then thecombination may be combined with the third component, etc.

Where animal (and particularly bovine) materials are used in the cultureof cells, they should be obtained from sources that are free fromtransmissible spongiform encaphalopathies (TSEs), and in particular freefrom bovine spongiform encephalopathy (BSE). Overall, it is preferred toculture cells in the total absence of animal-derived materials.

Where a compound is administered to the body as part of a compositionthen that compound may alternatively be replaced by a suitable pro-drug.

Where a cell substrate is used for reassortment or reverse geneticsprocedures, it is preferably one that has been approved for use in humanvaccine production, e.g., as in European Pharmacopoeia general chapter5.2.3., “Cell substrates for production of vaccines for human use.”

Accordingly, the present invention encompasses, but is not limited to,the following embodiments:

1. A method for enhancing an immune response in a subject, the methodcomprising the steps of:

selecting a subject that is immunocompromised or at risk ofimmunosuppression; and,

administering to the subject a vaccine comprising an adjuvant, ahigh-dose antigen, or combination thereof in an amount effective toenhance an immune response to the vaccine in the subject.

2. A method for enhancing an immune response, the method comprising thesteps of:

administering to a subject a vaccine comprising an adjuvant, a high-doseantigen, or combination thereof in an amount effective to enhance animmune response to the vaccine in the subject,

wherein the subject is immunocompromised or at risk ofimmunosuppression.

3. A method for administering a vaccine to a subject receiving animmunomodulatory therapy in an amount effective to elicit a protectiveimmune response to the vaccine antigen(s).4. A method for treating an immunocompromised subject comprising a stepof administering to the subject a pharmaceutical composition in anamount effective to elicit an immune response in the subject.5. A composition for use in a method for enhancing an immune response ina subject receiving an immunomodulatory therapy in an amount effectiveto enhance an immune response in the subject.6. A vaccine composition for a patient on an immunomodulatory therapy.7. A vaccine composition for use in a patient on an immunomodulatorytherapy.8. A vaccine composition for prevention of an infection in a patient onan immunomodulatory therapy.9. A composition for use as a medicament for treating a subject who isimmunocompromised.10. Use of a composition for the manufacture of a medicament for raisingan immune response in a subject who is immunocompromised.11. A method for manufacturing an adjuvanted and/or high-dose vaccine,wherein the vaccine is for use in a subject who is immunocompromised.12. The composition, method, or use of any one of the precedingembodiments, wherein the subject has a condition associated withcompromised immunity.13. The composition, method, or use of any one of the precedingembodiments, wherein the subject is on a statin therapy, an NSAIDtherapy, or combination thereof.14. The composition, method, or use of any one of the precedingembodiment, wherein the subject is:

65 years or older;

60 years or older;

45 years or older;

between the age of 45 and 64;

between the age of 18 and 64; or,

an infant.

15. The composition, method, or use of any one of the precedingembodiment, wherein the subject has a disease or disorder associatedwith impaired immunity.16. The composition, method, or use of any one of the precedingembodiment, wherein the subject is on an immunomodulatory therapy.17. The composition, method, or use of any one of the precedingembodiment, wherein the subject has been on the therapy for at least 1week.18. The composition, method, or use of any one of the precedingembodiment, wherein the subject has been on the therapy for at least 2weeks, at least 3 weeks, at least 4 weeks, or longer.18. The composition, method, or use of any one of the precedingembodiment, wherein the subject is not currently on a therapy but was ona therapy which terminated within the last 3 months.19. The composition, method, or use of any one of the precedingembodiment, wherein the subject is not currently on a therapy but isscheduled to be on a therapy in the next 3 months.20. The composition, method, or use of any one of the precedingembodiment, wherein the subject is on a statin therapy, an NSAIDtherapy, an interferon therapy, an antipsychotic and/or antidepressanttherapy, or any combinations thereof.21. The composition, method, or use of any one of the precedingembodiment, wherein the statin therapy comprises a synthetic statin, anon-synthetic statin, or combination thereof.22. The composition, method, or use of any one of the precedingembodiment, wherein the statin therapy comprises a statin selected fromthe group consisting of:

Pravastatin, Simvastatin, Lovastatin and Mevastatin, Fluvastatin,Atorvastatin, Cerivastatin, Rosuvastatin and Pitavastatin.

23. The composition, method, or use of any one of the precedingembodiment, wherein the statin therapy comprises a synthetic statinselected from the group consisting of:

Fluvastatin, Atorvastatin, Cerivastatin, Rosuvastatin and Pitavastatin.

24. The composition, method, or use of any one of the precedingembodiment, wherein the NSAID therapy comprises one or more of thefollowing:

Salicylates (e.g., Aspirin (acetylsalicylic acid), Diflunisal(Dolobid™), Salsalate (Disalcid™) and Choline Magnesium Trisalicylate(Trilisate™)); Propionic acid derivatives (e.g., Ibuprofen,Dexibuprofen, Naproxen, Fenoprofen, Ketoprofen, Dexketoprofen,Flurbiprofen, Oxaprozin and Loxoprofen); Acetic acid derivatives (e.g.,Indomethacin, Tolmetin, Sulindac, Etodolac, Ketorolac, Diclofenac,Aceclofenac and Nabumetone); Enolic acid (Oxicam) derivatives (e.g.,Piroxicam, Meloxicam, Tenoxicam, Droxicam, Lornoxicam and Isoxicam);Anthranilic acid derivatives (Fenamates) (e.g., Mefenamic acid,Meclofenamic acid, Flufenamic acid and Tolfenamic acid); Selective COX-2inhibitors (Coxibs) (e.g., Celecoxib, Rofecoxib, Valdecoxib, Parecoxib,Lumiracoxib, Etoricoxib and Firocoxib); Sulfonanilides (e.g.,Nimesulide) and others, such as Licofelone, H-harpagide and Lysineclonixinate.

25. The composition, method, or use of any one of the precedingembodiment, wherein the composition comprises an adjuvant.26. The composition, method, or use of any one of the precedingembodiment, wherein the composition comprises a surfactant.27. The composition, method, or use of any one of the precedingembodiment, wherein the composition comprises an oil-in-water emulsion.28. The composition, method, or use of any one of the precedingembodiment, wherein the composition comprises an aluminum salt adjuvant.29. The composition, method, or use of any one of the precedingembodiment, wherein the composition comprises an aluminum phosphateadjuvant and/or an aluminum hydroxide adjuvant.30. The composition, method, or use of any one of the precedingembodiment, wherein the composition comprises a TLR agonist.31. The composition, method, or use of any one of the precedingembodiment, wherein the composition comprises virosomes.32. The composition, method, or use of any one of the precedingembodiment, wherein the composition comprises squalene.33. The composition, method, or use of any one of the precedingembodiment, wherein the composition comprises a polysorbate.34. The composition, method, or use of any one of the precedingembodiment, wherein the composition comprises squalene, polysorbate 80,and sorbitan trioleate.35. The composition, method, or use of any one of the precedingembodiment, wherein the composition comprises about 4.3% squalene, about0.5% polysorbate 80 and about 0.48% sorbitan trioleate by weight.36. The composition, method, or use of any one of the precedingembodiment, wherein the composition is or comprises a vaccine.37. The composition, method, or use of any one of the precedingembodiment, wherein the composition is or comprises an antigen.38. The composition, method, or use of any one of the precedingembodiment, wherein the composition is or comprises a high-dose antigen,a standard-dose antigen, or a low-dose antigen.39. The composition, method, or use of any one of the precedingembodiment, wherein the composition comprises between a ⅛ and a ten-foldthe amount of a standard-dose antigen.40. The composition, method, or use of any one of the precedingembodiment, wherein the composition comprises between a two-fold and aten-fold the amount of a standard-dose antigen.41. The composition, method, or use of any one of the precedingembodiment, wherein the composition does not contain an adjuvant.42. The composition, method, or use of any one of the precedingembodiment, wherein the composition does not contain an oil-in-wateremulsion adjuvant.43. The composition, method, or use of any one of the precedingembodiment, wherein the composition is an influenza vaccine.44. The composition, method, or use of any one of the precedingembodiment, wherein the composition is a multivalent influenza vaccine.45. The composition, method, or use of any one of the precedingembodiment, wherein the influenza vaccine comprises between about 30 μgand about 150 μg of antigen per strain.46. The composition, method, or use of any one of the precedingembodiment, wherein the influenza vaccine comprises about 60 μg ofantigen per strain.47. The composition, method, or use of any one of the precedingembodiment, wherein the influenza vaccine comprises an H1N1 strain, anH3N2 strain, a B strain, or any combination thereof.

EXEMPLIFICATION

As mentioned above, statin therapy has been associated with secondaryeffects on the immune system. These effects include immunomodulatory andanti-inflammatory effects. Since many patients who routinely takestatins are elderly and the elderly are at higher risk of thecomplications of influenza (Thompson, W. W., Shay, D. K., Weintraub, E.,Brammer, L., Cox, N., Anderson, L. J., & Fukuda, K. (2003). Mortalityassociated with influenza and respiratory syncytial virus in the UnitedStates. Jama, 289(2): 179-186), we utilized data from a largecomparative immunogenicity study of adjuvanted and unadjuvantedinfluenza vaccines in the elderly to evaluate the influence of statintherapy on the immune response to influenza vaccine.

We utilized the immunogenicity measurements available from a comparativetrial of adjuvanted versus unadjuvanted influenza vaccine in patients toevaluate the influence of statin therapy on the immune response tovaccination. Overall, data on more than 5,000 trial participants wereavailable for analysis. Comparison of HAI geometric mean titers toinfluenza H1N1, H3N2 and B strains in individuals on and off chronicstatin therapy revealed that titers in statin recipients were 38%, 67%and 38%, respectively, lower in statin recipients than in individualsnot on statins. This immunosuppressive effect of statins on vaccineimmune response was particularly dramatic in individuals on syntheticstatins. These effects were seen in both the adjuvanted and unadjuvantedvaccine groups. However, since titers in the adjuvanted vaccine groupwere higher, the impact of statin therapy on vaccine response was atleast partially counteracted by the use of adjuvanted vaccine. Theseresults have implications both for future clinical trial design as wellas for vaccine usage recommendations in the elderly.

Methods

During the influenza seasons of 2009-2010 and 2010-2011, a randomized,controlled, observer-blind clinical trial was conducted comparing thesafety and immunogenicity of MF-59 adjuvanted trivalent influenzavaccine (“aTIV”) and unadjuvanted TIV in over 14,000 adults older than65 years of age in Colombia, Panama, the Philippines and the USA (Frey SE, Aplasca-De Los Reyes M R, Reynales H, Bermal N N, Nicolay U,Narasimhan V, Forleo-Neto E, and Arora A K. Comparison of the Safety andImmunogenicity of an MF-59 adjuvanted with a Non-adjuvanted seasonalinfluenza vaccine in Elderly Subjects. Vaccine in press). As part ofthis evaluation, information on statin use was collected and this wasconsidered as a potential confounder in comparative analyses.

Blood samples were obtained on the day of vaccination and 28 daysfollowing receipt of seasonally appropriate influenza vaccine. HAItiters were determined using standard methodology (Murphy, B. R.,Phelan, M. A., Nelson, D. L., Yarchoan, R., Tierney, E. L., Alling, D.W., & Chanock, R. M. (1981). Hemagglutinin-specific enzyme-linkedimmunosorbent assay for antibodies to influenza A and B viruses. Journalof clinical microbiology, 13(3): 554-560).

For the purposes of our current post-hoc analysis, patients wereclassified as being on statin therapy if they had been taking medicationfor 28 days or more prior to receipt of vaccination and through day 22after vaccination. Individuals who had not received statins during thistime window were considered to be controls. The small number ofindividuals that did not fit into either group were dropped from theanalysis. Patients were further stratified as to whether they were onsynthetic or natural occurring statins.

Geometric Mean Titers (GMT) and GMT ratios were then compared betweenthe statin and control groups against the vaccine homologous influenza Astrains H1N1 (California), H3N2 (Perth), and influenza B (Brisbane). Inadjusted comparisons, an ANCOVA analysis included the followingvariables: vaccine group (aTIV, TIV), statin user in days −28 to day 22(yes/no), high risk status (yes/no), sex, log-pre-vaccination titer andage (both continuous variables). Because an evaluation of theinteraction between vaccine type and statins did not reveal an impact ofthe type of vaccine on the impact of statins on the immune response(p>0.05), ratios of the GMTs in statin recipients and controls werecalculated for the combined study group.

Results

A total of 6961 subjects, 3479 in the MF-59 adjuvanted TIV group(“aTIV”) and 3482 in the unadjuvanted TIV group (“TIV”), had day 22 HAItiters available for analysis. Overall, 2798 and 2786 aTIV and TIVrecipients respectively were controls and 681 and 696 individualsrespectively were determined to meet the definition of being statinusers. Of the statin users, 76% of aTIV and 74% of TIV statin users weretaking fermentation derived statins (Pravastatin, Simvastatin,Lovastatin, Adivocor) and the remainder were taking synthetic statins(Fluvastatin, Atorvastatin, Rosuvastatin). Overall, 75% of statin userswere considered to have a high risk medical condition. The most commoncategory was underlying neurologic disease followed by COPD, asthma,congestive heart failure, renal insufficiency and hepatic disease.(Table 1). Overall, 55% of statin users and 68% of controls were maleand 74% of controls and 67% of statin users were between 65-75 years ofage with the remainder being older than 75 years of age. The results ofHAI GMTs against the three influenza vaccine strains are shown in TablesTwo with the day one ratio being adjusted for age, risk group andvaccine and the day 22 ratio also adjusted for pre-titer.

TABLE 1 Co-morbidities in each group On Statins day No Statins −22 today 28 aTIV TIV aTIV TIV Co-morbidity (N = 2798) (N = 2786) (N = 681) (N= 696) Asthma 120 (4%)  112 (4%)  42 (6%) 43 (6%) CHF 46 (2%) 46 (2%) 31(5%) 33 (5%) COPD 105 (4%)  113 (4%)   66 (10%) 61 (9%) Hepatic disease 9 (<1%)  9 (<1%)  4 (<1%)  4 (<1%) Neurological . . . 591 (21%) 576(21%) 485 (71%) 469 (67%) Renal insufficiency 28 (1%)  27 (<1%) 21 (3%)30 (4%)

TABLE 2 GMT Titer by Statin Use and Vaccine Group for each of threeinfluenza strains Influenza No Statin Group Statin Group Strain Day aTIVTIV aTIV TIV H1N1 1 n 2797 2784 681 696 California GMT 12 12 22 24 0995% CI 12-13 11-12 19-24 21-26 Ratio 0.62 (0.57-0.67) ST−/ST+ (95% CI)22 n 2797 2786 681 696 GMT 196 140 170 129 95% CI 185-206 133-148155-188 117-142 Ratio 1.38 (1.27-1.50) ST−/ST+ (95% CI) H3N2 Perth 1 n2797 2784 681 696 09 GMT 50 49 47 44 95% CI 47-53 46-52 42-52 40-49Ratio 1.13 (1.02-1.25) ST−/ST+ (95% CI) 22 n 2797 2785 681 696 GMT 669421 356 209 95% CI 638-701 402-441 324-392 190-230 Ratio 1.67(1.54-1.80) ST−/ST+ (95% CI) B Brisbane 08 1 n 2798 2786 681 696 GMT 9.69.4 16 17 95% CI 9.2-9.9 9.1-9.8 15-18 15-18 Ratio 0.68 (0.64-0.72)ST−/ST+ (95% CI) 22 n 2798 2786 681 696 GMT 50 43 48 40 95% CI 47-5241-46 44-52 37-44 Ratio 1.38 (1.28-1.49) ST−/ST+ (95% CI)

As can be seen in Table Two, overall pre-titers were equal in eachvaccine group for each of the three strains. However, individuals onstatins had higher pre-titers against H1N1 and influenza B whereas thereverse was true for influenza H3N2. Post vaccination day 22 titers weresignificantly higher in aTIV recipients for all three antigens. Inanalyses comparing statin recipients with controls regardless of vaccinetype, the GMT titer ratio was 38% higher in controls for H1N1, 67%higher for H3N2 and 38% higher for influenza B indicating a markedreduction in immunogenicity in statin recipients for all three antigensdespite adjustment for age, high risk group status, pre-titer and typeof vaccine received. Similar data was obtained when testing againstheterologous strains of influenza B (Malaysia), and H3N2 (Brisbane andWisconsin) (data not shown).

Results stratified by statin type are shown in Table 3.

TABLE 3 Influence of Statin Type on Vaccine Response as assessed at day22 Ratio ST −/ Ratio ST −/ Ratio Fermentation Synthetic Fermentation/ST + ST + Synthetic Homologous Strain (95% CI) (95% CI) (95% CI) BBrisbane 08 1.32 (1.21-1.43) 1.59 (1.39-1.81) 1.21 (1.05-1.39) H1N1California 09 1.31 (1.20-1.44) 1.62 (1.40-1.87) 1.23 (1.05-1.44) H3N2Perth 09 1.59 (1.46-1.73) 1.91 (1.68-2.19) 1.20 (1.04-1.39)

Patients receiving fermentation derived statins had higher titers thanthose on synthetic statins indicating that the latter had a greaterimmunosuppressive effect on influenza vaccine response. Similarnon-statically significant trends were also seen in testing againstheterologous strains.

Discussion

Within developing countries, life expectancy has been steadilyincreasing with an increasing proportion of the populations indeveloping countries being elderly (Mathers, C. D., Sadana, R., Salomon,J. A., Murray, C. J., & Lopez, A. D. (2001). Healthy life expectancy in191 countries, 1999. The Lancet, 357(9269): 1685-1691). Suchimprovements have been attributed to public health measures includingvaccinations and medications such as statins(www.who.int/bulletin/volumes/86/2/07-040089/en/ accessed Jul. 16,2014). Statins are widely used in adults and the elderly for treatmentof hypercholesterolemia. We have shown that this class of drugs,especially synthetically derived statins, dramatically suppress theimmune response to both adjuvanted and unadjuvanted influenza vaccinesin the population evaluated. Surprisingly, for two of the three strains,adjuvanted vaccine was able to at least counteract thisimmunosuppressive effect. For the H3N2 strain tested, while adjuvantedvaccine titers were higher than those for TIV, titers in statinrecipients were lower following aTIV than in controls receivingunadjuvanted vaccine.

Given the increasingly complex nature of health interventions inhigh-risk populations, including the elderly and those on certainmedications, long-term therapy in particular, it will be important toassess potential interactions between such interventions.

Our results stand in direct contrast to studies of the impact of statinson vaccine immune response to hepatitis A vaccine and tetanus toxoid inyoung adults. In a study by Seigrist et al., the mean age of subjectswas 24 years. In this study healthy subjects were randomized to receiveatorvastin or placebo. Response to hepatitis A vaccine was assessed 28days following receipt of vaccine. No difference in the immune responsewas seen between the two groups. It is important to note, however, thatin contrast to our study in which statin recipients were on chronictherapy at the time of vaccination, in this hepatitis A study, studyparticipants did not begin statin therapy until the day of vaccination(Packard R S, Schlegel S, Senouf D, Burger F, Sigaud P, Perneger T,Seigrist C A, and Mach F. Atorvastin Treatment and Vaccination Efficacy.J Clin Pharmacology 2007, 47:1022-1027). In another study, Brantly et alevaluated response to tetanus toxoid in healthy volunteers. Similar tothe hepatitis A study, healthy study participants were randomized toreceive atorvastin or placebo and began medication on the day ofvaccination. Surprisingly, study participants in this study assigned tothe statin group had three fold higher anti-TT IgG levels (Lee P Y,Sumpia P O, Byars J A, Kelly K M, Zhuang H, Shuster J S, Theriaque D W,Segal M S, Reeves W H, and Brantly M L. Short-term atorvastin treatmentenhances specific antibody production following tetanus toxoidvaccination in healthy volunteers. Vaccine 2006, 24: 4035-4040). The twoclear differences between these studies and the results we report hereare the much older age of our study group and the fact that in thetetanus toxoid and hepatitis A studies, participants had not beenchronically exposed to statins at the time of vaccination. It is ofcourse possible that one or both of these factors contribute to thecontrasting study results. However, since most statin users take themedication long term, the results of these two studies have limitedutility to evaluating the influence of routine statin therapy.

Studies of influenza vaccine effectiveness in the elderly have revealedsuboptimal levels of effectiveness. In a study by Monto in elderlynursing home patients, vaccination was 33% against influenza likeillness and 43% against pneumonia (Monto, A. S., Hornbuckle, K., &Ohmit, S. E. (2001). Influenza vaccine effectiveness among elderlynursing home residents: a cohort study. American journal ofepidemiology, 154(2), 155-160). In an earlier meta-analysis study usingdata from twenty observational studies largely conducted between 1970sand 1980s, Gross found higher pooled estimates of vaccine efficacy of56% (95% CI, 39% to 68%) for prevention respiratory illness, 53% (CI,35% to 66%) for prevention of pneumonia, 50% (CI, 28% to 65%) forprevention of hospitalization, and 68% (CI, 56% to 76%) for preventingdeath (Gross, P. A., Hermogenes, A. W., Sacks, H. S., Lau, J., &Levandowski, R. A. (1995). The efficacy of influenza vaccine in elderlypersonsA meta-analysis and review of the literature. Annals of Internalmedicine, 123(7), 518-527). Of interest is that estimates of efficacyagainst respiratory illness and pneumonia in the earlier years whenstatin use was less common are higher than those in the more recentstudy. In light of analyses disclosed here, it is also possible thatthese differences are due to different influenza strains and otherpopulation factors.

Statins have been considered as adjunct agents in the preventionpneumonia because their immunosuppressive effect might lower baselineinflammatory status and thus the severity of pneumonia. Observationalstudies of statin use in COPD have reported reductions in mortality of30%-50% following pneumonia or infective exacerbations in statin users(Young R, Hopkins R J. Statin Use in Pneumonia. The American Journal ofMedicine, Volume 126, Issue 7, e11-e12). Other studies have not found animpact of statins on pneumonia and sepsis risk (Yende S, Milbrandt E B,Kellum J A, Kong L, Delude R L, Weissfeld L A and Angus D C.Understanding the potential role of statins in pneumonia and sepsis.Critical Care Medicine 2011, 39(8): 1871-1878). Fedson has recommendedconsideration of statins as therapeutic agents in the treatment ofpneumonia in the elderly (Fedson, D. S. (2013). Treating influenza withstatins and other immunomodulatory agents. Antiviral research, 99(3),417-435). In a commentary he states that while system biologists havesuggested the use of immunomodulatory agents such as statins in thetreatment of influenza, randomized clinical trials of this approachshould precede their routine use of this. Fedson similarly points outthat especially in pandemics where severe disease may proceed vaccineavailability by many months, consideration should be given to evaluatingstatins as potential agents to reduce inflammation and hence severity ofdisease (Fedson, D. S. (2013). How will physicians respond to the nextinfluenza pandemic?. Clinical infectious diseases, cit695).

Clearly the impact of statins on the immune system and consequentvaccine response as well as disease risk are complex. While theimmunosuppressive effects of statins may be desirable in the acutedisease state, the same effect can be deleterious when it impactsvaccine response. We have shown a dramatic effect of long term statinuse on the immune response to influenza vaccine in the testedpopulation. This negative effect should be taken into account whenevaluating the immunogenicity and effectiveness of influenza vaccinesaffected population and potentially in considering preferential use ofadjuvanted vaccines and/or high-dose vaccines such populations ofsubjects to counteract drug-induced immunosuppression.

The various features and embodiments of the present invention, referredto in individual sections above apply, as appropriate, to othersections, mutatis mutandis. Consequently features specified in onesection may be combined with features specified in other sections, asappropriate.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1: A method for enhancing an immune response in a subject comprisingadministering a vaccine, wherein the vaccine comprises: (a) an adjuvant;(b) a high-dose antigen; or a combination of (a) and (b); and whereinthe subject is under the age of 65 and on a statin therapy. 2: Themethod of claim 1, wherein the adjuvant is an aluminum salt adjuvant oran oil-in-water emulsion adjuvant. 3: The method of claim 2, wherein theoil-in-water emulsion adjuvant comprises squalene. 4: The method ofclaim 2, wherein the oil-in-water adjuvant comprises squalene,polysorbate 80, and sorbitan trioleate. 5: The method of claim 1,wherein the vaccine comprises between a ⅛ and a ten-fold the amount of astandard dose antigen. 6: The method of claim 1, wherein the vaccine isan influenza vaccine. 7: The method of claim 5, wherein the influenzavaccine comprises between about 30 μg and about 150 μg of antigen perstrain. 8: The method of claim 6, wherein the influenza vaccinecomprises an H1N1 strain, an H3N2 strain, a B strain, or any combinationthereof. 9: The method of claim 1, wherein the vaccine does not containan oil-in-water emulsion adjuvant. 10: The method of claim 1, whereinthe subject is: a) currently on a statin therapy; b) not currently on astatin therapy but was on a statin therapy which terminated within thelast 3 months; or c) not currently on a statin therapy but is scheduledto be on a statin therapy in the next 3 months. 11: The method of claim1, wherein the subject is between the age of 60 and
 64. 12: The methodof claim 1, wherein the subject has a disease or disorder associatedwith impaired immunity. 13: The method of claim 1, wherein the statintherapy comprises a synthetic statin, a non-synthetic statin, or acombination thereof. 14: The method of claim 13, wherein the statintherapy comprises a synthetic statin selected from the group consistingof: Fluvastatin, Atorvastatin, Cerivastatin, Rosuvastatin andPitavastatin. 15: The method of claim 3, wherein the oil-in-wateremulsion adjuvant further comprises a surfactant. 16: The method ofclaim 4, wherein the oil-in-water adjuvant comprises about 4.3%squalene, about 0.5% polysorbate 80 and about 0.48% sorbitan trioleateby weight. 17: The method of claim 5, wherein the vaccine comprisesbetween a two-fold and a ten-fold the amount of a standard dose antigen.18: The method of claim 6, wherein the influenza vaccine is amultivalent influenza vaccine. 19: The method of claim 7, wherein theinfluenza vaccine comprises about 60 μg of antigen per strain. 20: Themethod of claim 6, wherein the influenza vaccine comprises between about30 μg and about 150 μg of antigen per strain. 21: The method of claim20, wherein the influenza vaccine comprises about 60 μg of antigen perstrain. 22: The method of claim 7, wherein the influenza vaccinecomprises an H1N1 strain, an H3N2 strain, a B strain, or any combinationthereof. 23: The method of claim 10, wherein the subject has been on thestatin therapy for at least 1 week. 24: The method of claim 23, whereinthe subject has been on the statin therapy for at least 2 weeks. 25: Themethod of claim 24, wherein the subject has been on the statin therapyfor at least 3 weeks. 26: The method of claim 25, wherein the subjecthas been on the statin therapy for at least 4 weeks or longer. 27: Themethod of claim 13, wherein the statin therapy comprises a statinselected from the group consisting of: Pravastatin, Simvastatin,Lovastatin and Mevastatin, Fluvastatin, Atorvastatin, Cerivastatin,Rosuvastatin and Pitavastatin.